Chimeric antigen receptors targeting b-cell maturation antigen

ABSTRACT

The invention provides CARs (CARs) that specifically bind to BCMA (B-Cell Maturation Antigen). The invention further relates to engineered immune cells comprising such CARs, CAR-encoding nucleic acids, and methods of making such CARs, engineered immune cells, and nucleic acids. The invention further relates to therapeutic methods for use of these CARs and engineered immune cells for the treatment of a condition associated with malignant cells expressing BCMA (e.g., cancer).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/085,317, filed on Mar. 30, 2016, which claims the benefit of U.S.Provisional Application No. 62/146,825 filed Apr. 13, 2015, U.S.Provisional Application No. 62/286,473 filed Jan. 25, 2016, and U.S.Provisional Application No. 62/301,177 filed Feb. 29, 2016, all of whichare hereby incorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includesan electronically submitted sequence listing in .txt format. The .txtfile contains a sequence listing entitled“ALGN_003_04US_SeqList_ST25.txt” created on Jan. 29, 2019, and having asize of ˜372,605 bytes. The sequence listing contained in this .txt fileis part of the specification and is incorporated herein by reference inits entirety.

Field

The invention relates to chimeric antigen receptors (CAR). CARs are ableto redirect immune cell specificity and reactivity toward a selectedtarget exploiting the ligand-binding domain properties. In particular,the invention relates to CARs that specifically bind to B-CellMaturation Antigen (BCMA specific CARs). The invention further relatesto polynucleotides encoding BCMA specific CAR and isolated cellsexpressing BCMA specific CARs at their surface. The invention furtherrelates to methods for engineering immune cells expressing BCMA specificCARs at their surface. The invention is particularly useful for thetreatment of B-cell lymphomas and leukemia. The invention furtherrelates to immune cells comprising the BCMA specific CARs (BCMA specificCAR-T cells), compositions comprising the BCMA specific CAR-T cells, andmethods of using the BCMA specific CAR-T cells for treating conditionsassociated with malignant cells expressing BCMA (e.g., cancer).

Background

Multiple myeloma is a malignancy characterized by an accumulation ofclonal plasma cells (see, e.g., Lonial et al., Clinical Cancer Res.,77(6): 1264-1277 (2011)). Current therapies for MM often causeremissions, but nearly all patients eventually relapse and die (see,e.g., Rajkumar, Nature Rev. Clinical Oncol, 5(8): 479-491 (2011)).

Adoptive transfer of T cells genetically modified to recognizemalignancy-associated antigens is showing promise as a new approach totreating cancer (see, e.g., Brenner et al., Current Opinion inImmunology, 22(2): 251-257 (2010); Rosenberg et al., Nature ReviewsCancer, 8(4): 299-308 (2008)). T cells can be genetically modified toexpress chimeric antigen receptors (CARs), which are fusion proteinscomprised of an antigen recognition moiety and T cell activation domains(see, e.g., Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2): 720-724(1993), and Sadelain et al., Curr. Opin. Immunol, 21(2): 215-223(2009)).

B-cell maturation antigen (BCMA, CD269, or TNFRSF17) is a member of thetumor necrosis factor receptor (TNFR) superfamily. BCMA was identifiedin a malignant human T cell lymphoma containing a t(4;16) translocation.The gene is selectively expressed in the B-cell lineage with the highestexpression in plasmablasts and plasma cells, antibody secreting cells.BCMA binds two ligands, B-cell activation factor (BAFF) (also calledB-lymphoctye stimulator (BLyS) and APOL-related leukocyte expressedligand (TALL-1)) and a proliferation-inducing ligand (APRIL) withaffinity of 1 uM and 16 nM, respectively. Binding of APRIL or BAFF toBCMA promotes a signaling cascade involving NF-kappa B, Elk-1, c-JunN-terminal kinase and the p38 mitogen-activated protein kinase, whichproduce signals for cell survival and proliferation. BCMA is alsoexpressed on malignant B cells and several cancers that involve Blymphocytes including multiple myeloma, plasmacytoma, Hodgkin'sLymphoma, and chronic lymphocytic leukemia. In autoimmune diseases whereplasmablasts are involved such as systemic lupus erythematosus (SLE) andrheumatoid arthritis, BCMA expressing antibody-producing cells secreteautoantibodies that attack self. In the case of multiple myeloma, about24,000 new cases are newly diagnosed in the United States each year, andthis number represents about 15% of the newly diagnosed hematologicalcancers in the United States. An average of 11,000 deaths result frommultiple myeloma each year, and the average 5-year survival rate isabout 44%, with median survival of 50-55 months. Current treatment formultiple myeloma is focused on plasma cells apoptosis and/or decreasingosteoclast activity (e.g., chemotherapy, thalidomide, lenalidomide,bisphosphonates, and/or proteasome inhibitors such as bortezomib(VELCADE®) or carfilzomib). However, multiple myeloma remains anincurable disease, and almost all patients have developed resistance tothese agents and eventually relapse. Accordingly, an alternativetreatment to multiple myeloma, such as using an anti-BCMA antagonistincluding BCMA specific CARs and BCMA specific CAR-T cells, would make asuperior therapeutic agent.

SUMMARY

Chimeric antigen receptors (CARs) that bind to BCMA are provided. It isdemonstrated that certain BCMA specific CARs are effective whenexpressed in T cells to activate T cells upon contact with BCMA.Advantageously, the BCMA specific CARs provided herein bind human andcynomolgous monkey BCMA. Also advantageously, the BCMA specific CAR-Tcells provided herein exhibit degranulation activity, increasedinterferon gamma production, and/or cytotoxic activity upon contact withBCMA-expressing cells.

In one aspect, the invention provides a BCMA specific CAR comprising anextracellular ligand-binding domain, a first transmembrane domain, andan intracellular signaling domain, wherein the extracellularligand-binding domain domain comprises (a) a heavy chain variable (VH)region comprising (i) a VH complementarity determining region one (CDR1)comprising the sequence SYX₁MX₂, wherein X₁ is A or P; and X₂ is T, N,or S (SEQ ID NO: 301), GFTFX₁SY, wherein X₁ is G or S (SEQ ID NO: 302),or GFTFX₁SYX₂MX₃, wherein X₁ is G or S, X₂ is A or P; and X₃ is T, N, orS (SEQ ID NO: 303); (ii) a VH CDR2 comprising the sequenceAX₁X₂X₃X₄GX₅X₆X₇X₈YADX₉X₁₀KG, wherein X₁ is I, V, T, H, L, A, or C; X₂is S, D, G, T, I, L, F, M, or V; X₃ is G, Y, L, H, D, A, S, or M; X₄ isS, Q, T, A, F, or W; X₅ is G or T;X₆ is N, S, P, Y, W, or F; X₇ is S, T,I, L, T, A, R, V, K, G, or C; X₈ is F, Y, P, W, H, or G; X₉ is V, R, orL; and X₁₀ is G or T (SEQ ID NO: 305), or X₁X₂X₃X₄X₅X₆, wherein X₁ is S,V, I, D, G, T, L, F, or M; X₂ is G, Y, L, H, D, A, S, or M; X₃ is S, G,F, or W; X₄ is G or S; X₅ is G or T; and X₆ is N, S, P, Y, or W (SEQ IDNO: 306); and iii) a VH CDR3 comprising the sequence VSPIX₁X₂X₃X₄,wherein X₁ is A or Y; X₂ is A or S; and X₃ is G, Q, L, P, or E (SEQ IDNO: 307), or YWPMX₁X₂, wherein X₁ is D, S, T, or A; and X₂ is I, S, L,P, or D (SEQ ID NO: 308); and/or (b) a light chain variable (VL) regioncomprising (i) a VL CDR1 comprising the sequenceX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂, wherein X₁ is R, G, W, A, or C; X₂ is A, P,G, L, C, or S; X₃ is S, G, or R; X₄ is Q, C, E, V, or I; X₅ is S, L, P,G, A, R, or D; X₆ is V, G, or I; X₇ is S, E, D, or P; X₈ is S, P, F, A,M, E, V, N, D, or Y; X₉ is I, T, V, E, S, A, M, Q, Y, H, or R; X₁₀ is Yor F; X₁₁ is L, W, or P; and X₁₂ is A, S, or G (SEQ ID NO: 309); (ii) aVL CDR2 comprising the sequence X₁ASX₂RAX₃, wherein X₁ is G or D; X₂ isS or I; and X₃ is T or P (SEQ ID NO: 310); and (iii) a VL CDR3comprising the sequence QQYX₁X₂X₃PX₄T, wherein X₁ is G, Q, E, L, F, A,S, M, K, R, or Y; X₂ is S, R, T, G, V, F, Y, D, A, H, V, E, K, or C; X₃is W, F, or S; and X₄ is L or I (SEQ ID NO: 311), or QQYX₁X₂X₃PX₄,wherein X₁ is G, Q, E, L, F, A, S, M, R, K, or Y; X₂ is S, R, T, G, R,V, D, A, H, E, K, C, F, or Y; X₃ is W, S, or F; and X₄ is L or I (SEQ IDNO: 312).

In another aspect, the invention provides a BCMA specific CAR comprisingan extracellular ligand-binding domain, a first transmembrane domain,and an intracellular signaling domain, wherein the extracellular domaincomprises a single chain Fv fragment (scFv) comprising a heavy chainvariable (VH) region comprising three CDRs from the VH region comprisingthe sequence shown in SEQ ID NO: 33, 72, 39, 76, 83, 92, 25, or 8; and alight chain variable (VL) region comprising three CDRs from the VLregion shown in SEQ ID NO: 34, 73, 40, 77, 84, 93, 18, or 80. In someembodiments, the VH region can comprise the sequence shown in SEQ ID NO:33, 72, 39, 76, 83, 92, 25, or 8, or a variant thereof with one orseveral conservative amino acid substitutions in residues that are notwithin a CDR and/or the VL region can comprise the amino acid sequenceshown in SEQ ID NO: 34, 73, 40, 77, 84, 93, 18, or 80, or a variantthereof with one or several amino acid substitutions in amino acids thatare not within a CDR.

In some embodiments, the extracellular ligand-binding domain domain of aBCMA specific CAR provided herein comprises (a) a heavy chain variable(VH) region comprising (i) a VH complementarity determining region one(CDR1) comprising the sequence shown in SEQ ID NO: 150, 151, 152, 156,157, 129, 130, or 131; (ii) a VH CDR2 comprising the sequence shown in153, 154, 187, 188, 165, 166, 162, 159, 190, 191, 169, 154, 139, 140,132, or 133; and (iii) a VH CD3 comprising the sequence shown in 155,161, 134, or 137; and/or (b) a light chain variable region (VL)comprising (i) a VL CDR1 comprising the sequence shown in SEQ ID NO:209, 249, 226, 251, 262, 271, 217, or 377; (ii) a VL CDR2 comprising thesequence shown in SEQ ID NO: 221, 252, or 210; and (iii) a VL CDR3comprising the sequence shown in SEQ ID NO: 222, 225, 227, 253, 263,272, 216, or 214.

In some embodiments, the extracellular ligand-binding domain domain of aBCMA specific CAR provided herein comprises (a) a heavy chain variable(VH) region comprising (i) a VH complementarity determining region one(CDR1) comprising the sequence shown in SEQ ID NO: 150, 151, or 152;(ii) a VH CDR2 comprising the sequence shown in 153 or 154; and (iii) aVH CD3 comprising the sequence shown in 155; and/or (b) a light chainvariable region (VL) comprising (i) a VL CDR1 comprising the sequenceshown in SEQ ID NO: 209; (ii) a VL CDR2 comprising the sequence shown inSEQ ID NO: 221, and (iii) a VL CDR2 comprising the sequence shown in SEQID NO: 222.

In some embodiments, the extracellular ligand-binding domain domain of aBCMA specific CAR provided herein comprises (a) a heavy chain variable(VH) region comprising (i) a VH complementarity determining region one(CDR1) comprising the sequence shown in SEQ ID NO: 150, 151, or 152;(ii) a VH CDR2 comprising the sequence shown in 187 or 188; and (iii) aVH CD3 comprising the sequence shown in 155; and/or (b) a light chainvariable region (VL) comprising (i) a VL CDR1 comprising the sequenceshown in SEQ ID NO: 249; (ii) a VL CDR2 comprising the sequence shown inSEQ ID NO: 221, and (iii) a VL CDR3 comprising the sequence shown in SEQID NO: 225.

In some embodiments, the extracellular ligand-binding domain domain of aBCMA specific CAR provided herein comprises (a) a heavy chain variable(VH) region comprising (i) a VH complementarity determining region one(CDR1) comprising the sequence shown in SEQ ID NO: 150, 151, or 152;(ii) a VH CDR2 comprising the sequence shown in 165 or 166; and (iii) aVH CD3 comprising the sequence shown in 155; and/or (b) a light chainvariable region (VL) comprising (i) a VL CDR1 comprising the sequenceshown in SEQ ID NO: 226; (ii) a VL CDR2 comprising the sequence shown inSEQ ID NO: 221, and (iii) a VL CDR3 comprising the sequence shown in SEQID NO: 227.

In some embodiments, the extracellular ligand-binding domain domain of aBCMA specific CAR provided herein comprises (a) a heavy chain variable(VH) region comprising (i) a VH complementarity determining region one(CDR1) comprising the sequence shown in SEQ ID NO: 156, 151, or 157;(ii) a VH CDR2 comprising the sequence shown in 162 or 159; and (iii) aVH CD3 comprising the sequence shown in 161; and/or (b) a light chainvariable region (VL) comprising (i) a VL CDR1 comprising the sequenceshown in SEQ ID NO: 251; (ii) a VL CDR2 comprising the sequence shown inSEQ ID NO: 252, and (iii) a VL CDR3 comprising the sequence shown in SEQID NO: 253.

In some embodiments, the extracellular ligand-binding domain domain of aBCMA specific CAR provided herein comprises (a) a heavy chain variable(VH) region comprising (i) a VH complementarity determining region one(CDR1) comprising the sequence shown in SEQ ID NO: 156, 151, or 157;(ii) a VH CDR2 comprising the sequence shown in 190 or 191; and (iii) aVH CD3 comprising the sequence shown in 161; and/or (b) a light chainvariable region (VL) comprising (i) a VL CDR1 comprising the sequenceshown in SEQ ID NO: 262; (ii) a VL CDR2 comprising the sequence shown inSEQ ID NO: 252, and (iii) a VL CDR3 comprising the sequence shown in SEQID NO: 263.

In some embodiments, the extracellular ligand-binding domain domain of aBCMA specific CAR provided herein comprises (a) a heavy chain variable(VH) region comprising (i) a VH complementarity determining region one(CDR1) comprising the sequence shown in SEQ ID NO: 150, 151, or 152;(ii) a VH CDR2 comprising the sequence shown in 169 or 154; and (iii) aVH CD3 comprising the sequence shown in 155; and/or (b) a light chainvariable region (VL) comprising (i) a VL CDR1 comprising the sequenceshown in SEQ ID NO: 271; (ii) a VL CDR2 comprising the sequence shown inSEQ ID NO: 221, and (iii) a VL CDR3 comprising the sequence shown in SEQID NO: 272.

In some embodiments, the extracellular ligand-binding domain domain of aBCMA specific CAR provided herein comprises (a) a heavy chain variable(VH) region comprising (i) a VH complementarity determining region one(CDR1) comprising the sequence shown in SEQ ID NO: 129, 130, or 131;(ii) a VH CDR2 comprising the sequence shown in 139 or 140; and (iii) aVH CD3 comprising the sequence shown in 134; and/or (b) a light chainvariable region (VL) comprising (i) a VL CDR1 comprising the sequenceshown in SEQ ID NO: 217; (ii) a VL CDR2 comprising the sequence shown inSEQ ID NO: 210, and (iii) a VL CDR3 comprising the sequence shown in SEQID NO: 216.

In some embodiments, the extracellular ligand-binding domain domain of aBCMA specific CAR provided herein comprises (a) a heavy chain variable(VH) region comprising (i) a VH complementarity determining region one(CDR1) comprising the sequence shown in SEQ ID NO: 129, 130, or 131;(ii) a VH CDR2 comprising the sequence shown in 132 or 133; and (iii) aVH CD3 comprising the sequence shown in 137; and/or (b) a light chainvariable region (VL) comprising (i) a VL CDR1 comprising the sequenceshown in SEQ ID NO: 377; (ii) a VL CDR2 comprising the sequence shown inSEQ ID NO: 210, and (iii) a VL CDR3 comprising the sequence shown in SEQID NO: 214.

In some embodiments, the intracellular signaling domain comprises a CD3ζsignalling domain. In some embodiments, the intracellular signalingdomain comprises a 4-1BB domain. In some embodiments, the CAR canfurther comprise another intracellular signaling domain. In someembodiments, the additional intracellular signaling domain can comprisea 4-1BB domain.

In some embodiments, the CAR can comprise a stalk domain between theextracellular ligand-binding domain and the first transmembrane domain.In some embodiments, the stalk domain can be selected from the groupconsisting of: a human CD8α hinge, an IgG1 hinge, and an FcγRIIIα hinge.

In some embodiments, the first transmembrane domain can comprise a CD8αchain transmembrane domain.

In some embodiments, the CAR can comprise a CD20 epitope.

In some embodiments, the CAR can comprise another extracellularligand-binding domain which is not specific for BCMA.

In some embodiments, the BCMA specific CAR can comprise the amino acidsequence shown in SEQ ID NO: 396.

In some embodiments of a CAR, the extracellular ligand-bindingdomain(s), the first transmembrane domain, and intracellular signalingdomain(s) are on a single polypeptide.

In some embodiments, the CAR can comprise a second transmembrane domain,wherein the first transmembrane domain and the extracellularligand-binding domain(s) are on a first polypeptide, and wherein thesecond transmembrane domain and the intracellular signaling domain(s)are on a second polypeptide, wherein the first transmembrane domaincomprises a transmembrane domain from the a chain of the high-affinityIgE receptor (FcεRI) and the second transmembrane domain comprises atransmembrane domain from the γ or β chain of FcεRI. In someembodiments, the CAR can comprise a third polypeptide comprising a thirdtransmembrane domain fused to an intracellular signaling domain from aco-stimulatory molecule, wherein the third transmembrane domaincomprises a transmembrane domain from the γ or β chain of FcεRI.

In another aspect, the invention provides an isolated polynucleotidecomprising a nucleic acid sequence encoding a BCMA specific CAR asdescribed herein.

In another aspect, the invention provides an expression vectorcomprising a nucleic acid sequence encoding a BCMA specific CAR antibodyas described herein.

In another aspect, the invention provides engineered immune cellexpressing at its cell surface membrane a BCMA specific CAR as describedherein. In some embodiments, the engineered immunce cell can compriseanother CAR which is not specific for BCMA. In some embodiments, theengineered immunce cell can comprise a polynucleotide encoding a suicidepolypeptide. In some embodiments, the suicide polypeptide is RQR8.

In some embodiments, the immune cell can be derived from an inflammatoryT-lymphocyte, a cytotoxic T-lymphocyte, a regulatory T-lymphocyte, or ahelper T-lymphocyte.

In some embodiments, the engineered immune cell can comprise adisruption one or more endogenous genes, wherein the endogenous geneencodes TCRα, TCRβ, CD52, glucocorticoid receptor (GR), deoxycytidinekinase (DCK), or an immune checkpoint protein such as for exampleprogrammed death-1 (PD-1).

In some embodiments, immune cell is obtained from a healthy donor. Insome embodiments, the immune cell is obtained from a patient.

In another aspect, the invention provides an engineered immune cellexpressing at its cell surface membrane a BCMA specific CAR as describedherein for use as a medicament. In some embodiments, the medicament isfor use in treatment of a B-cell related cancer selecting from the groupconsisting of multiple myeloma, malignant plasma cell neoplasm,Hodgkin's lymphoma, nodular lymphocyte predominant Hodgkin's lymphoma,Kahler's disease and Myelomatosis, plasma cell leukemia, plasmacytoma,B-cell prolymphocytic leukemia, hairy cell leukemia, B-cellnon-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), chroniclymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronicmyeloid leukemia (CML), follicular lymphoma, Burkitt's lymphoma,marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma,precursor B-lymphoblastic lymphoma, myeloid leukemia, Waldenstrom'smacroglobulienemia, diffuse large B cell lymphoma, follicular lymphoma,marginal zone lymphoma, mucosa-associated lymphatic tissue lymphoma,small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma,primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmactyiclymphoma, Waldenström macroglobulinemia, nodal marginal zone B celllymphoma, splenic marginal zone lymphoma, intravascular large B-celllymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, Tcell/histiocyte-rich large B-cell lymphoma, primary central nervoussystem lymphoma, primary cutaneous diffuse large B-cell lymphoma (legtype), EBV positive diffuse large B-cell lymphoma of the elderly,diffuse large B-cell lymphoma associated with inflammation,intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma,plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associatedmulticentric Castleman disease, B-cell lymphoma unclassified withfeatures intermediate between diffuse large B-cell lymphoma and Burkittlymphoma, B-cell lymphoma unclassified with features intermediatebetween diffuse large B-cell lymphoma and classical Hodgkin lymphoma,and other B-cell related lymphoma.

In another aspect, the invention provides a method of engineering animmune cell comprising: providing an immune cell; and expressing at thesurface of the cell at least one BCMA specific CAR as described herein.

In some embodiments, the method comprises: providing an immune cell;introducing into the cell at least one polynucleotide encoding said BCMAspecific CAR; and expressing said polynucleotide into the cell.

In some embodiments, the method comprises providing an immune cell;introducing into the cell at least one polynucleotide encoding said BCMAspecific CAR; and introducing at least one other CAR which is notspecific for BCMA.

In another aspect, the invention provides a method of treating a subjectsuffering from a condition associated with malignant cells, the methodcomprising: providing a immune cell expressing at the surface a BCMAspecific CAR as described herein; and administering said immune cells tosaid patient.

In another aspect, the invention provides a pharmaceutical compositioncomprising an engineered immune cell as described herein.

In another aspect, the invention provides a method of treating acondition associated with malignant cells expressing BCMA in a subjectcomprising administering to a subject in need thereof an effectiveamount of a pharmaceutical composition of claim comprising an engineeredimmune cell as described herein. In some embodiments, the condition is acancer. In some embodiments, the cancer is a B-cell related cancerselecting from the group consisting of multiple myeloma, malignantplasma cell neoplasm, Hodgkin's lymphoma, nodular lymphocyte predominantHodgkin's lymphoma, Kahler's disease and Myelomatosis, plasma cellleukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cellleukemia, B-cell non-Hodgkin's lymphoma (NHL), acute myeloid leukemia(AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia(ALL), chronic myeloid leukemia (CML), follicular lymphoma, Burkitt'slymphoma, marginal zone lymphoma, mantle cell lymphoma, large celllymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia,Waldenstrom's macroglobulienemia, diffuse large B cell lymphoma,follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphatictissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma,Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma,lymphoplasmactyic lymphoma, Waldenström macroglobulinemia, nodalmarginal zone B cell lymphoma, splenic marginal zone lymphoma,intravascular large B-cell lymphoma, primary effusion lymphoma,lymphomatoid granulomatosis, T cell/histiocyte-rich large B-celllymphoma, primary central nervous system lymphoma, primary cutaneousdiffuse large B-cell lymphoma (leg type), EBV positive diffuse largeB-cell lymphoma of the elderly, diffuse large B-cell lymphoma associatedwith inflammation, intravascular large B-cell lymphoma, ALK-positivelarge B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphomaarising in HHV8-associated multicentric Castleman disease, B-celllymphoma unclassified with features intermediate between diffuse largeB-cell lymphoma and Burkitt lymphoma, B-cell lymphoma unclassified withfeatures intermediate between diffuse large B-cell lymphoma andclassical Hodgkin lymphoma, and other B-cell related lymphoma.

In another aspect, the invention provides a method of inhibiting tumorgrowth or progression in a subject who has malignant cells expressingBCMA, comprising administering to the subject in need thereof aneffective amount of a pharmaceutical composition comprising anengineered immune cell as described herein.

In another aspect, the invention provides a method inhibiting metastasisof malignant cells expressing BCMA in a subject, comprisingadministering to the subject in need thereof an effective amount of thepharmaceutical composition comprising an engineered immune cell asdescribed herein.

In another aspect, the invention provides a method inducing tumorregression in a subject who has malignant cells expressing BCMA,comprising administering to the subject in need thereof an effectiveamount of the pharmaceutical composition of a pharmaceutical compositioncomprising an engineered immune cell as described herein.

In some embodiments, any of the above methods further comprisesadministering one or more additional therapies, such as for example, amonoclonal antibody and/or a chemotherapeutic. In some embodiments, themonoclonal antibody can be, for example, an antibody that binds to acheckpoint inhibitor such as, for example, an anti-PD-1 antibody or ananti-PD-L1 antibody. In some embodiments, any of the above methodsfurther comprises administering a nucleoside analog therapy, such as forexample fludarabine or clofarabine, to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a graph summarizing the results of treatment with BCMAspecific CAR-T cells in the MM1.S tumor model.

FIG. 2 depicts a graph summarizing the results of treatment with BCMAspecific CAR-T cells in the Molp8 tumor model.

DETAILED DESCRIPTION

The invention disclosed herein provides chimeric antigen receptors(CARs) and immune cells comprising CARs (CAR-T cells) that specificallybind to BCMA (e.g., human BCMA). The invention also providespolynucleotides encoding these CARs, compositions comprising these CAR-Tcells, and methods of making and using these CARs and CAR-T cells. Theinvention also provides methods for treating a condition associated withmalignant BCMA expression in a subject, such as cancer.

General Techniques

The practice of the invention will employ, unless otherwise indicated,conventional techniques of molecular biology (including recombinanttechniques), microbiology, cell biology, biochemistry and immunology,which are within the skill of the art. Such techniques are explainedfully in the literature, such as, Molecular Cloning: A LaboratoryManual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press;Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in MolecularBiology, Humana Press; Cell Biology: A Laboratory Notebook (J. E.Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney,ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: LaboratoryProcedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds.,1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press,Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D.Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practicalapproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (ColdSpring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

Definitions

The term “extracellular ligand-binding domain” as used herein refers toan oligo- or polypeptide that is capable of binding a ligand.Preferably, the domain will be capable of interacting with a cellsurface molecule. For example, the extracellular ligand-binding domainmay be chosen to recognize a ligand that acts as a cell surface markeron target cells associated with a particular disease state.

The term “stalk domain” or “hinge domain” are used interchangeablyherein to refer to any oligo- or polypeptide that functions to link thetransmembrane domain to the extracellular ligand-binding domain. Inparticular, stalk domains are used to provide more flexibility andaccessibility for the extracellular ligand-binding domain.

The term “intracellular signaling domain” refers to the portion of aprotein which transduces the effector signal function signal and directsthe cell to perform a specialized function.

A “co-stimulatory molecule” as used herein refers to the cognate bindingpartner on a T cell that specifically binds with a co-stimulatoryligand, thereby mediating a co-stimulatory response by the cell, suchas, but not limited to proliferation. Co-stimulatory molecules include,but are not limited to an MHC class I molecule, BTLA and Toll ligandreceptor. Examples of costimulatory molecules include CD27, CD28, CD8,4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 anda ligand that specifically binds with CD83 and the like.

A “co-stimulatory ligand” refers to a molecule on an antigen presentingcell that specifically binds a cognate co-stimulatory signal molecule ona T cell, thereby providing a signal which, in addition to the primarysignal provided by, for instance, binding of a TCR/CD3 complex with anMHC molecule loaded with peptide, mediates a T cell response, including,but not limited to, proliferation activation, differentiation and thelike. A co-stimulatory ligand can include but is not limited to CD7,B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, induciblecostimulatory igand (ICOS-L), intercellular adhesion molecule (ICAM,CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin βreceptor, 3/TR6, ILT3, ILT4, an agonist or antibody that binds Tollligand receptor and a ligand that specifically binds with B7-H3. Aco-stimulatory ligand also encompasses, inter alia, an antibody thatspecifically binds with a co-stimulatory molecule present on a T cell,such as but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1,ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT,NKG2C, B7-H3, a ligand that specifically binds with CD83.

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule. As used herein, the termencompasses not only intact polyclonal or monoclonal antibodies, butalso fragments thereof (such as Fab, Fab′, F(ab′)₂, Fv), single chain(scFv) and domain antibodies (including, for example, shark and camelidantibodies), and fusion proteins comprising an antibody, and any othermodified configuration of the immunoglobulin molecule that comprises anantigen recognition site. An antibody includes an antibody of any class,such as IgG, IgA, or IgM (or sub-class thereof), and the antibody neednot be of any particular class. Depending on the antibody amino acidsequence of the constant region of its heavy chains, immunoglobulins canbe assigned to different classes. There are five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these maybe further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2. The heavy-chain constant regions that correspond tothe different classes of immunoglobulins are called alpha, delta,epsilon, gamma, and mu, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

The term “antigen binding fragment” or “antigen binding portion” of anantibody, as used herein, refers to one or more fragments of an intactantibody that retain the ability to specifically bind to a given antigen(e.g., BCMA). Antigen binding functions of an antibody can be performedby fragments of an intact antibody. Examples of binding fragmentsencompassed within the term “antigen binding fragment” of an antibodyinclude Fab; Fab′; F(ab′)₂; an Fd fragment consisting of the VH and CH1domains; an Fv fragment consisting of the VL and VH domains of a singlearm of an antibody; a single domain antibody (dAb) fragment (Ward etal., Nature 341:544-546, 1989), and an isolated complementaritydetermining region (CDR).

An antibody, an antibody conjugate, or a polypeptide that“preferentially binds” or “specifically binds” (used interchangeablyherein) to a target (e.g., BCMA protein) is a term well understood inthe art, and methods to determine such specific or preferential bindingare also well known in the art. A molecule is said to exhibit “specificbinding” or “preferential binding” if it reacts or associates morefrequently, more rapidly, with greater duration and/or with greateraffinity with a particular cell or substance than it does withalternative cells or substances. An antibody “specifically binds” or“preferentially binds” to a target if it binds with greater affinity,avidity, more readily, and/or with greater duration than it binds toother substances. For example, an antibody that specifically orpreferentially binds to a BCMA epitope is an antibody that binds thisepitope with greater affinity, avidity, more readily, and/or withgreater duration than it binds to other BCMA epitopes or non-BCMAepitopes. It is also understood that by reading this definition, forexample, an antibody (or moiety or epitope) that specifically orpreferentially binds to a first target may or may not specifically orpreferentially bind to a second target. As such, “specific binding” or“preferential binding” does not necessarily require (although it caninclude) exclusive binding. Generally, but not necessarily, reference tobinding means preferential binding.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. As known in the art, the variableregions of the heavy and light chain each consist of four frameworkregions (FR) connected by three complementarity determining regions(CDRs) also known as hypervariable regions. The CDRs in each chain areheld together in close proximity by the FRs and, with the CDRs from theother chain, contribute to the formation of the antigen binding site ofantibodies. There are at least two techniques for determining CDRs: (1)an approach based on cross-species sequence variability (i.e., Kabat etal. Sequences of Proteins of Immunological Interest, (5th ed., 1991,National Institutes of Health, Bethesda MD)); and (2) an approach basedon crystallographic studies of antigen-antibody complexes (Al-lazikaniet al., 1997, J. Molec. Biol. 273:927-948). As used herein, a CDR mayrefer to CDRs defined by either approach or by a combination of bothapproaches.

A “CDR” of a variable domain are amino acid residues within the variableregion that are identified in accordance with the definitions of theKabat, Chothia, the accumulation of both Kabat and Chothia, AbM,contact, and/or conformational definitions or any method of CDRdetermination well known in the art. Antibody CDRs may be identified asthe hypervariable regions originally defined by Kabat et al. See, e.g.,Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5thed., Public Health Service, NIH, Washington D.C. The positions of theCDRs may also be identified as the structural loop structures originallydescribed by Chothia and others. See, e.g., Chothia et al., Nature342:877-883, 1989. Other approaches to CDR identification include the“AbM definition,” which is a compromise between Kabat and Chothia and isderived using Oxford Molecular's AbM antibody modeling software (nowAccelrys®), or the “contact definition” of CDRs based on observedantigen contacts, set forth in MacCallum et al., J. Mol. Biol.,262:732-745, 1996. In another approach, referred to herein as the“conformational definition” of CDRs, the positions of the CDRs may beidentified as the residues that make enthalpic contributions to antigenbinding. See, e.g., Makabe et al., Journal of Biological Chemistry,283:1156-1166, 2008. Still other CDR boundary definitions may notstrictly follow one of the above approaches, but will nonethelessoverlap with at least a portion of the Kabat CDRs, although they may beshortened or lengthened in light of prediction or experimental findingsthat particular residues or groups of residues or even entire CDRs donot significantly impact antigen binding. As used herein, a CDR mayrefer to CDRs defined by any approach known in the art, includingcombinations of approaches. The methods used herein may utilize CDRsdefined according to any of these approaches. For any given embodimentcontaining more than one CDR, the CDRs may be defined in accordance withany of Kabat, Chothia, extended, AbM, contact, and/or conformationaldefinitions.

As used herein, “monoclonal antibody” refers to an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the invention may be made by the hybridoma method firstdescribed by Kohler and Milstein, Nature 256:495, 1975, or may be madeby recombinant DNA methods such as described in U.S. Pat. No. 4,816,567.The monoclonal antibodies may also be isolated from phage librariesgenerated using the techniques described in McCafferty et al., Nature348:552-554, 1990, for example.

As used herein, “humanized” antibody refers to forms of non-human (e.g.murine) antibodies that are chimeric immunoglobulins, immunoglobulinchains, or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen binding subsequences of antibodies) that contain minimalsequence derived from non-human immunoglobulin. Preferably, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a complementarity determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, or rabbit having the desiredspecificity, affinity, and capacity. In some instances, Fv frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, the humanized antibodymay comprise residues that are found neither in the recipient antibodynor in the imported CDR or framework sequences, but are included tofurther refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsowill comprise at least a portion of an immunoglobulin constant region ordomain (Fc), typically that of a human immunoglobulin. Preferred areantibodies having Fc regions modified as described in WO 99/58572. Otherforms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDRL3, CDR H1, CDR H2, or CDR H3) which are altered with respect to theoriginal antibody, which are also termed one or more CDRs “derived from”one or more CDRs from the original antibody.

As used herein, “human antibody” means an antibody having an amino acidsequence corresponding to that of an antibody produced by a human and/orwhich has been made using any of the techniques for making humanantibodies known to those skilled in the art or disclosed herein. Thisdefinition of a human antibody includes antibodies comprising at leastone human heavy chain polypeptide or at least one human light chainpolypeptide. One such example is an antibody comprising murine lightchain and human heavy chain polypeptides. Human antibodies can beproduced using various techniques known in the art. In one embodiment,the human antibody is selected from a phage library, where that phagelibrary expresses human antibodies (Vaughan et al., NatureBiotechnology, 14:309-314, 1996; Sheets et al., Proc. Natl. Acad. Sci.(USA) 95:6157-6162, 1998; Hoogenboom and Winter, J. Mol. Biol., 227:381,1991; Marks et al., J. Mol. Biol., 222:581, 1991). Human antibodies canalso be made by immunization of animals into which human immunoglobulinloci have been transgenically introduced in place of the endogenousloci, e.g., mice in which the endogenous immunoglobulin genes have beenpartially or completely inactivated. This approach is described in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and5,661,016. Alternatively, the human antibody may be prepared byimmortalizing human B lymphocytes that produce an antibody directedagainst a target antigen (such B lymphocytes may be recovered from anindividual or from single cell cloning of the cDNA, or may have beenimmunized in vitro). See, e.g., Cole et al. Monoclonal Antibodies andCancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al., J. Immunol.,147 (1):86-95, 1991; and U.S. Pat. No. 5,750,373.

The term “chimeric antibody” is intended to refer to antibodies in whichthe variable region sequences are derived from one species and theconstant region sequences are derived from another species, such as anantibody in which the variable region sequences are derived from a mouseantibody and the constant region sequences are derived from a humanantibody.

The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” areused interchangeably herein to refer to chains of amino acids of anylength, preferably, relatively short (e.g., 10-100 amino acids). Thechain may be linear or branched, it may comprise modified amino acids,and/or may be interrupted by non-amino acids. The terms also encompassan amino acid chain that has been modified naturally or by intervention;for example, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labeling component. Also included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),as well as other modifications known in the art. It is understood thatthe polypeptides can occur as single chains or associated chains.

A “monovalent antibody” comprises one antigen binding site per molecule(e.g., IgG or Fab). In some instances, a monovalent antibody can havemore than one antigen binding sites, but the binding sites are fromdifferent antigens.

A “bivalent antibody” comprises two antigen binding sites per molecule(e.g., IgG). In some instances, the two binding sites have the sameantigen specificities. However, bivalent antibodies may be bispecific.

A “bispecific,” “dual-specific” or “bifunctional” antibody is a hybridantibody having two different antigen binding sites. The two antigenbinding sites of a bispecific antibody bind to two different epitopes,which may reside on the same or different protein targets.

Antibodies of the invention can be produced using techniques well knownin the art, e.g., recombinant technologies, phage display technologies,synthetic technologies or combinations of such technologies or othertechnologies readily known in the art (see, for example, Jayasena, S.D., Clin. Chem., 45: 1628-50, 1999 and Fellouse, F. A., et al, J. Mol.Biol., 373(4):924-40, 2007).

As known in the art, “polynucleotide,” or “nucleic acid,” as usedinterchangeably herein, refer to chains of nucleotides of any length,and include DNA and RNA. The nucleotides can be deoxyribonucleotides,ribonucleotides, modified nucleotides or bases, and/or their analogs, orany substrate that can be incorporated into a chain by DNA or RNApolymerase. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thechain. The sequence of nucleotides may be interrupted by non-nucleotidecomponents. A polynucleotide may be further modified afterpolymerization, such as by conjugation with a labeling component. Othertypes of modifications include, for example, “caps”, substitution of oneor more of the naturally occurring nucleotides with an analog,internucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.), those containingalkylators, those with modified linkages (e.g., alpha anomeric nucleicacids, etc.), as well as unmodified forms of the polynucleotide(s).Further, any of the hydroxyl groups ordinarily present in the sugars maybe replaced, for example, by phosphonate groups, phosphate groups,protected by standard protecting groups, or activated to prepareadditional linkages to additional nucleotides, or may be conjugated tosolid supports. The 5′ and 3′ terminal OH can be phosphorylated orsubstituted with amines or organic capping group moieties of from 1 to20 carbon atoms. Other hydroxyls may also be derivatized to standardprotecting groups. Polynucleotides can also contain analogous forms ofribose or deoxyribose sugars that are generally known in the art,including, for example, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or2′-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomericsugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranosesugars, furanose sugars, sedoheptuloses, acyclic analogs and abasicnucleoside analogs such as methyl riboside. One or more phosphodiesterlinkages may be replaced by alternative linking groups. Thesealternative linking groups include, but are not limited to, embodimentswherein phosphate is replaced by P(O)S(“thioate”), P(S)S (“dithioate”),(O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂ (“formacetal”), in whicheach R or R′ is independently H or substituted or unsubstituted alkyl(1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl,cycloalkyl, cycloalkenyl or araldyl. Not all linkages in apolynucleotide need be identical. The preceding description applies toall polynucleotides referred to herein, including RNA and DNA.

As known in the art a “constant region” of an antibody refers to theconstant region of the antibody light chain or the constant region ofthe antibody heavy chain, either alone or in combination.

As used herein, “substantially pure” refers to material which is atleast 50% pure (i.e., free from contaminants), more preferably, at least90% pure, more preferably, at least 95% pure, yet more preferably, atleast 98% pure, and most preferably, at least 99% pure.

A “host cell” includes an individual cell or cell culture that can be orhas been a recipient for vector(s) for incorporation of polynucleotideinserts. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation. A host cell includes cellstransfected in vivo with a polynucleotide(s) of this invention.

As used herein, “immune cell” refers to a cell of hematopoietic originfunctionally involved in the initiation and/or execution of innateand/or adaptative immune response.

As known in the art, the term “Fc region” is used to define a C-terminalregion of an immunoglobulin heavy chain. The “Fc region” may be a nativesequence Fc region or a variant Fc region. Although the boundaries ofthe Fc region of an immunoglobulin heavy chain might vary, the human IgGheavy chain Fc region is usually defined to stretch from an amino acidresidue at position Cys226, or from Pro230, to the carboxyl-terminusthereof. The numbering of the residues in the Fc region is that of theEU index as in Kabat. Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991. The Fc region of animmunoglobulin generally comprises two constant regions, CH2 and CH3.

As used in the art, “Fc receptor” and “FcR” describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and FcγRIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. FcRs are reviewed in Ravetch and Kinet,Ann. Rev. Immunol., 9:457-92, 1991; Capel et al., Immunomethods,4:25-34, 1994; and de Haas et al., J. Lab. Clin. Med., 126:330-41, 1995.“FcR” also includes the neonatal receptor, FcRn, which is responsiblefor the transfer of maternal IgGs to the fetus (Guyer et al., J.Immunol., 117:587, 1976; and Kim et al., J. Immunol., 24:249, 1994).

The term “compete”, as used herein with regard to an antibody, meansthat a first antibody, or an antigen binding fragment (or portion)thereof, binds to an epitope in a manner sufficiently similar to thebinding of a second antibody, or an antigen binding portion thereof,such that the result of binding of the first antibody with its cognateepitope is detectably decreased in the presence of the second antibodycompared to the binding of the first antibody in the absence of thesecond antibody. The alternative, where the binding of the secondantibody to its epitope is also detectably decreased in the presence ofthe first antibody, can, but need not be the case. That is, a firstantibody can inhibit the binding of a second antibody to its epitopewithout that second antibody inhibiting the binding of the firstantibody to its respective epitope. However, where each antibodydetectably inhibits the binding of the other antibody with its cognateepitope or ligand, whether to the same, greater, or lesser extent, theantibodies are said to “cross-compete” with each other for binding oftheir respective epitope(s). Both competing and cross-competingantibodies are encompassed by the invention. Regardless of the mechanismby which such competition or cross-competition occurs (e.g., sterichindrance, conformational change, or binding to a common epitope, orportion thereof), the skilled artisan would appreciate, based upon theteachings provided herein, that such competing and/or cross-competingantibodies are encompassed and can be useful for the methods disclosedherein.

As used herein “autologous” means that cells, a cell line, or populationof cells used for treating patients are originating from said patient orfrom a Human Leucocyte Antigen (HLA) compatible donor.

As used herein “allogeneic” means that cells or population of cells usedfor treating patients are not originating from said patient but from adonor.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, one or more ofthe following: reducing the proliferation of (or destroying) neoplasticor cancerous cells, inhibiting metastasis of neoplastic cells, shrinkingor decreasing the size of BCMA expressing tumor, remission of a BCMAassociated disease (e.g., cancer), decreasing symptoms resulting from aBCMA associated disease (e.g., cancer), increasing the quality of lifeof those suffering from a BCMA associated disease (e.g., cancer),decreasing the dose of other medications required to treat a BCMAassociated disease (e.g., cancer), delaying the progression of a BCMAassociated disease (e.g., cancer), curing a BCMA associated disease(e.g., cancer), and/or prolong survival of patients having a BCMAassociated disease (e.g., cancer).

“Ameliorating” means a lessening or improvement of one or more symptomsas compared to not administering a BCMA antibody or a BCMA antibodyconjugate. “Ameliorating” also includes shortening or reduction induration of a symptom.

As used herein, an “effective dosage” or “effective amount” of drug,compound, or pharmaceutical composition is an amount sufficient toeffect any one or more beneficial or desired results. For prophylacticuse, beneficial or desired results include eliminating or reducing therisk, lessening the severity, or delaying the outset of the disease,including biochemical, histological and/or behavioral symptoms of thedisease, its complications and intermediate pathological phenotypespresenting during development of the disease. For therapeutic use,beneficial or desired results include clinical results such as reducingincidence or amelioration of one or more symptoms of various BCMAassociated diseases or conditions (such as for example multiplemyeloma), decreasing the dose of other medications required to treat thedisease, enhancing the effect of another medication, and/or delaying theprogression of the BCMA associated disease of patients. An effectivedosage can be administered in one or more administrations. For purposesof this invention, an effective dosage of drug, compound, orpharmaceutical composition is an amount sufficient to accomplishprophylactic or therapeutic treatment either directly or indirectly. Asis understood in the clinical context, an effective dosage of a drug,compound, or pharmaceutical composition may or may not be achieved inconjunction with another drug, compound, or pharmaceutical composition.Thus, an “effective dosage” may be considered in the context ofadministering one or more therapeutic agents, and a single agent may beconsidered to be given in an effective amount if, in conjunction withone or more other agents, a desirable result may be or is achieved.

An “individual” or a “subject” is a mammal, more preferably, a human.Mammals also include, but are not limited to, farm animals, sportanimals, pets, primates, horses, dogs, cats, mice and rats.

As used herein, “vector” means a construct, which is capable ofdelivering, and, preferably, expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid or phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, DNA or RNA expressionvectors encapsulated in liposomes, and certain eukaryotic cells, such asproducer cells.

As used herein, “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid. An expressioncontrol sequence can be a promoter, such as a constitutive or aninducible promoter, or an enhancer. The expression control sequence isoperably linked to the nucleic acid sequence to be transcribed.

As used herein, “pharmaceutically acceptable carrier” or “pharmaceuticalacceptable excipient” includes any material which, when combined with anactive ingredient, allows the ingredient to retain biological activityand is non-reactive with the subject's immune system. Examples include,but are not limited to, any of the standard pharmaceutical carriers suchas a phosphate buffered saline solution, water, emulsions such asoil/water emulsion, and various types of wetting agents. Preferreddiluents for aerosol or parenteral administration are phosphate bufferedsaline (PBS) or normal (0.9%) saline. Compositions comprising suchcarriers are formulated by well known conventional methods (see, forexample, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro,ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Scienceand Practice of Pharmacy 21st Ed. Mack Publishing, 2005).

The term “k_(on)”, as used herein, refers to the rate constant forassociation of an antibody to an antigen.

The term “k_(off)”, as used herein, refers to the rate constant fordissociation of an antibody from the antibody/antigen complex.

The term “K_(D)”, as used herein, refers to the equilibrium dissociationconstant of an antibody-antigen interaction.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.” Numeric ranges are inclusive of the numbers defining the range.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the inventionencompasses not only the entire group listed as a whole, but each memberof the group individually and all possible subgroups of the main group,but also the main group absent one or more of the group members. Theinvention also envisages the explicit exclusion of one or more of any ofthe group members in the claimed invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent specification, including definitions, will control. Throughoutthis specification and claims, the word “comprise,” or variations suchas “comprises” or “comprising” will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers. Unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular.

Exemplary methods and materials are described herein, although methodsand materials similar or equivalent to those described herein can alsobe used in the practice or testing of the invention. The materials,methods, and examples are illustrative only and not intended to belimiting.

BCMA Specific CARs and Methods of Making Thereof

The invention provides CARs that bind to BCMA (e.g., human BCMA (e.g.,SEQ ID NO: 354 or accession number: Q02223-2). BCMA specific CARsprovided herein include single chain CARS and multichain CARs. The CARshave the ability to redirect T cell specificity and reactivity towardBCMA in a non-MHC-restricted manner, exploiting the antigen-bindingproperties of monoclonal antibodies. The non-MHC-restricted antigenrecognition gives T cells expressing CARs the ability to recognize anantigen independent of antigen processing, thus bypassing a majormechanism of tumor escape.

In some embodiments, CARs provided herein comprise an extracellularligand-binding domain (e.g., a single chain variable fragment (scFv)), atransmembrane domain, and an intracellular signaling domain. In someembodiments, the extracellular ligand-binding domain, transmembranedomain, and intracellular signaling domain are in one polypeptide, i.e.,in a single chain. Multichain CARs and polypeptides are also providedherein. In some embodiments, the mulitchain CARs comprise: a firstpolypeptide comprising a transmembrane domain and at least oneextracellular ligand-binding domain, and a second polypeptide comprisinga transmembrane domain and at least one intracellular signaling domain,wherein the polypeptides assemble together to form a multichain CAR.

In some embodiments, a BCMA specific multichain CAR is based on the highaffinity receptor for IgE (FcεRI). The FcεRI expressed on mast cells andbasophiles triggers allergic reactions. FcεRI is a tetrameric complexcomposed of a single a subunit, a single β subunit, and twodisulfide-linked γ subunits. The a subunit contains the IgE-bindingdomain. The β and γ subunits contain ITAMs that mediate signaltransduction. In some embodiments, the extracellular domain of the FcRαchain is deleted and replaced by a BCMA specific extracellularligand-binding domain. In some embodiments, the multichain BCMA specificCAR comprises an scFv that binds specifically to BCMA, the CD8α hinge,and the ITAM of the FcRβ chain. In some embodiments, the CAR may or maynot comprise the FcRγ chain.

In some embodiments, the extracellular ligand-binding domain comprisesan scFv comprising the light chain variable (VL) region and the heavychain variable (VH) region of a target antigen specific monoclonalantibody joined by a flexible linker. Single chain variable regionfragments are made by linking light and/or heavy chain variable regionsby using a short linking peptide (Bird et al., Science 242:423-426,1988). An example of a linking peptide is the GS linker having the aminoacid sequence (GGGGS)₃ (SEQ ID NO: 333), which bridges approximately 3.5nm between the carboxy terminus of one variable region and the aminoterminus of the other variable region. Linkers of other sequences havebeen designed and used (Bird et al., 1988, supra). In general, linkerscan be short, flexible polypeptides and preferably comprised of about 20or fewer amino acid residues. Linkers can in turn be modified foradditional functions, such as attachment of drugs or attachment to solidsupports. The single chain variants can be produced either recombinantlyor synthetically. For synthetic production of scFv, an automatedsynthesizer can be used. For recombinant production of scFv, a suitableplasmid containing polynucleotide that encodes the scFv can beintroduced into a suitable host cell, either eukaryotic, such as yeast,plant, insect or mammalian cells, or prokaryotic, such as E. coli.Polynucleotides encoding the scFv of interest can be made by routinemanipulations such as ligation of polynucleotides. The resultant scFvcan be isolated using standard protein purification techniques known inthe art.

In some embodiments, the extracellular ligand-binding domain comprises(a) a VH region comprising (i) a VH complementarity determining regionone (CDR1) comprising the sequence SYX₁MX₂, wherein X₁ is A or P; and X₂is T, N, or S (SEQ ID NO: 301), GFTFX₁SY, wherein X₁ is G or S (SEQ IDNO: 302), or GFTFX₁SYX₂MX₃, wherein X₁ is G or S, X₂ is A or P; and X₃is T, N, or S (SEQ ID NO: 303); (ii) a VH CDR2 comprising the sequenceAX₁X₂X₃X₄GX₅X₆X₇X₈YADX₉X₁₀KG, wherein X₁ is I, V, T, H, L, A, or C; X₂is S, D, G, T, I, L, F, M, or V; X₃ is G, Y, L, H, D, A, S, or M; X₄ isS, Q, T, A, F, or W; X₅ is G or T;X₆ is N, S, P, Y, W, or F; X₇ is S, T,I, L, T, A, R, V, K, G, or C; X₈ is F, Y, P, W, H, or G; X₉ is V, R, orL; and X₁₀ is G or T (SEQ ID NO: 305), or X₁X₂X₃X₄X₅X₆, wherein X₁ is S,V, I, D, G, T, L, F, or M; X₂ is G, Y, L, H, D, A, S, or M; X₃ is S, G,F, or W; X₄ is G or S; X₅ is G or T; and X₆ is N, S, P, Y, or W (SEQ IDNO: 306); and iii) a VH CDR3 comprising the sequence VSPIX₁X₂X₃X₄,wherein X₁ is A or Y; X₂ is A or S; and X₃ is G, Q, L, P, or E (SEQ IDNO: 307), or YWPMX₁X₂, wherein X₁ is D, S, T, or A; and X₂ is I, S, L,P, or D (SEQ ID NO: 308); and a VL region comprising (i) a VL CDR1comprising the sequence X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂, wherein X₁ is R, G,W, A, or C; X₂ is A, P, G, L, C, or S; X₃ is S, G, or R; X₄ is Q, C, E,V, or I; X₅ is S, L, P, G, A, R, or D; X₆ is V, G, or I; X₇ is S, E, D,or P; X₈ is S, P, F, A, M, E, V, N, D, or Y; X₉ is I, T, V, E, S, A, M,Q, Y, H, or R; X₁₀ is Y or F; X₁₁ is L, W, or P; and X₁₂ is A, S, or G(SEQ ID NO: 309); (ii) a VL CDR2 comprising the sequence X₁ASX₂RAX₃,wherein X₁ is G or D; X₂ is S or I; and X₃ is T or P (SEQ ID NO: 310);and (iii) a VL CDR3 comprising the sequence QQYX₁X₂X₃PX₄T, wherein X₁ isG, Q, E, L, F, A, S, M, K, R, or Y; X₂ is S, R, T, G, V, F, Y, D, A, H,V, E, K, or C; X₃ is W, F, or S; and X₄ is L or I (SEQ ID NO: 311), orQQYX₁X₂X₃PX₄, wherein X₁ is G, Q, E, L, F, A, S, M, R, K, or Y; X₂ is S,R, T, G, R, V, D, A, H, E, K, C, F, or Y; X₃ is W, S, or F; and X₄ is Lor I (SEQ ID NO: 312). In some embodiments, the VH and VL are linkedtogether by a flexible linker. In some embodiments a flexible linkercomprises the amino acid sequence shown in SEQ ID NO: 333.

In another aspect, provided is CAR, which specifically binds to BCMA,wherein the CAR comprises an extracellular ligand-binding domaincomprising: a VH region comprising a VH CDR1, VH CDR2, and VH CDR3 ofthe VH sequence shown in SEQ ID NO: 2, 3, 7, 8, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 35, 37, 39, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 83, 87, 92, 78, 95, 97, 99, 101, 104,106, 110, 112, 114, 76, 118, 120, 122, 112, 125, 127, 313, or 314;and/or a VL region comprising VL CDR1, VL CDR2, and VL CDR3 of the VLsequence shown in SEQ ID NO: 1, 4, 5, 6, 9, 10, 11, 12, 13, 15, 16, 17,18, 19, 20, 21, 22, 23, 34, 36, 38, 40, 41, 43, 45, 47, 49, 51, 53, 57,59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 317, 81, 82, 84, 85, 86, 88,89, 90, 91, 93, 94, 96, 98, 100, 102, 103, 105, 107, 108, 109, 111, 113,115, 116, 117, 119, 121, 123, 124, 126, 128, 315, or 316. In someembodiments, the VH and VL are linked together by a flexible linker. Insome embodiments a flexible linker comprises the amino acid sequenceshown in SEQ ID NO: 333.

In some embodiments, a CAR of the invention comprises an extracellularligand-binding domain having any one of partial light chain sequence aslisted in Table 1 and/or any one of partial heavy chain sequence aslisted in Table 1. In Table 1, the underlined sequences are CDRsequences according to Kabat and in bold according to Chothia, exceptfor the following heavy chain CDR2 sequences, in which the Chothia CDRsequences are underlined and the Kabat CDR sequences are in bold:P5A2_VHVL, A02_Rd4_0.6nM_C06, A02_Rd4_0.6nM_C09 A02_Rd4_6nM_C16,A02_Rd4_6nM_C03, A02_Rd4_6nM_C01, A02_Rd4_6nM_C26 A02_Rd4_6nM_C25,A02_Rd4_6nM_C22, A02_Rd4_6nM_C19, A02_Rd4_0.6nM_C03 A02_Rd4_6nM_C07,A02_Rd4_6nM_C23, A02_Rd4_0.6nM_C18, A02_Rd4_6nM_C10, A02_Rd4_6nM_C05,A02_Rd4_0.6nM_C10, A02_Rd4_6nM_C04, A02_Rd4_0.6nM_C26,A02_Rd4_0.6nM_C13, A02_Rd4_0.6nM_C01, A02_Rd4_6nM_C08, P5C1_VHVL,C01_Rd4_6nM_C24, C01_Rd4_6nM_C26, C01_Rd4_6nM_C10, C01_Rd4_0.6nM_C27,C01_Rd4_6nM_C20, C01_Rd4_6nM_C12, C01_Rd4_0.6nM_C16, C01_Rd4_0.6nM_C09,C01_Rd4_6nM_C09, C01_Rd4_0.6nM_C03, C01_Rd4_0.6nM_C06, C01_Rd4_6nM_C04,COMBO_Rd4_0.6nM_C22, COMBO_Rd4_6nM_C21, COMBO_Rd4_6nM_C10COMBO_Rd4_0.6nM_C04, COMBO_Rd4_6nM_C25, COMBO_Rd4_0.6nM_C21,COMBO_Rd4_6nM_C11, COMBO_Rd4_0.6nM_C20, COMBO_Rd4_6nM_C09,COMBO_Rd4_6nM_C08, COMBO_Rd4_0.6nM_C19, COMBO_Rd4_0.6nM_C02,COMBO_Rd4_0.6nM_C23, COMBO_Rd4_0.6nM_C29, COMBO_Rd4_0.6nM_C09,COMBO_Rd4_6nM_C12, COMBO_Rd4_0.6nM_C30, COMBO_Rd4_0.6nM_C14,COMBO_Rd4_6nM_C07, COMBO_Rd4_6nM_C02, COMBO_Rd4_0.6nM_C05,COMBO_Rd4_0.6nM_C17, COMBO_Rd4_6nM_C22, and COMBO_Rd4_0.6nM_C11.

TABLE 1 mAb Light Chain Heavy Chain P6E01/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA P6E01 ASQSVSSSYLAWYQQKPGQAPASGFTFGSYAMTWVRQAPGKGLE RLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAV YGSPPSFTFGQGTKVEIK (SEQYYCARVSPIASGMDYWGQGTLVT ID NO: 1) VSS (SEQ ID NO: 2) P6E01/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA H3.AQASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYGSPPSFTFGQGTKVEIK (SEQ YYCARVSPIAAQMDYWGQGTLVT ID NO: 1) VSS (SEQ IDNO: 3) L1.LGF/ EIVLTQSPGTLSLSPGERATLSC EVQLLESGGGLVQPGGSLRLSCA L3.KW/RASQSLGSFYLAWYQQKPGQA ASGFTFGSYAMTWVRQAPGKGLE P6E01PRLLIYGASSRATGIPDRFSGSG WVSAISGSGGNTFYADSVKGRFTISGTDFTLTISRLEPEDFAVYYCKH SRDNSKNTLYLQMNSLRAEDTAV YGWPPSFTFGQGTKVEIK (SEQYYCARVSPIASGMDYWGQGTLVT ID NO: 4) VSS (SEQ ID NO: 2) L1.LGF/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.NY/ASQSLGSFYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE P6E01RLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQHSRDNSKNTLYLQMNSLRAEDTAV YNYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVTID NO: 5) VSS (SEQ ID NO: 2) L1.GDF/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L3.NY/ ASQSVGDFYLAWYQQKPGQAPASGFTFGSYAMTWVRQAPGKGLE P6E01 RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYNYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVT ID NO: 6) VSS (SEQ IDNO: 2) L1.LGF/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.KW/ASQSLGSFYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.ALRLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCKHY SRDNSKNTLYLQMNSLRAEDTAV GWPPSFTFGQGTKVEIK (SEQID YYCARARVSPIAALMDYWGQGTL NO: 4) VTVSS (SEQ ID NO: 7) L1.LGF/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.KW/ASQSLGSFYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.APRLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCKHY SRDNSKNTLYLQMNSLRAEDTAV GWPPSFTFGQGTKVEIK (SEQID YYCARVSPIAAPMDYWGQGTLVT NO: 4) VSS (SEQ ID NO: 8) L1.LGF/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.KW/ASQSLGSFYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AQRLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCKHY SRDNSKNTLYLQMNSLRAEDTAV GWPPSFTFGQGTKVEIK (SEQID YYCARVSPIAAQMDYWGQGTLVT NO: 4) VSS (SEQ ID NO: 3) L1.LGF/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.PY/ASQSLGSFYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.APRLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIAAPMDYWGQGTLVTID NO: 9) VSS (SEQ ID NO: 8) L1.LGF/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L3.PY/ ASQSLGSFYLAWYQQKPGQAPASGFTFGSYAMTWVRQAPGKGLE H3.AQ RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIAAQMDYWGQGTLVT ID NO: 9) VSS (SEQ IDNO: 3) L1.LGF/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.NY/ASQSLGSFYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.ALRLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQHSRDNSKNTLYLQMNSLRAEDTAV YNYPPSFTFGQGTKVEIK (SEQ YYCARVSPIAALMDYWGQGTLVTID NO: 10) VSS (SEQ ID NO: 7) L1.LGF/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L3.NY/ ASQSLGSFYLAWYQQKPGQAPASGFTFGSYAMTWVRQAPGKGLE H3.AP RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYNYPPSFTFGQGTKVEIK (SEQ YYCARVSPIAAPMDYWGQGTLVT ID NO: 10) VSS (SEQ IDNO: 8) L1.LGF/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.NY/ASQSLGSFYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AQRLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQHSRDNSKNTLYLQMNSLRAEDTAV YNYPPSFTFGQGTKVEIK (SEQ YYCARVSPIAAQMDYWGQGTLVTID NO: 10) VSS (SEQ ID NO: 3) L1.GDF/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L3.KW/ ASQSVGDFYLAPWYQQKPGQAPASGFTFGSYAMTWVRQAPGKGLE H3.AL RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCKHYSRDNSKNTLYLQMNSLRAEDTAV GWPPSFTFGQGTKVEIK (SEQ IDYYCARVSPIAALMDYWGQGTLVT NO: 11) VSS (SEQ ID NO: 7) L1.GDF/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.KW/ASQSVGDFYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.APRLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCKHY SRDNSKNTLYLQMNSLRAEDTAV GWPPSFTFGQGTKVEIK (SEQID YYCARVSPIAAPMDYWGQGTLVT NO: 11) VSS (SEQ ID NO: 8) L1.GDF/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.KW/ASQSVGDFYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AQRLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCKHY SRDNSKNTLYLQMNSLRAEDTAV GWPPSFTFGQGTKVEIK (SEQID YYCARVSPIAAQMDYWGQGTLVT NO: 11) VSS (SEQ ID NO: 3) L1.GDF/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.PY/ASQSVGDFYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AQRLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIAAQMDYWGQGTLVTID NO: 12) VSS (SEQ ID NO: 3) L1.GDF/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L3.NY/ ASQSVGDFYLAWYQQKPGQAPASGFTFGSYAMTWVRQAPGKGLE H3.AL RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYNYPPSFTFGQGTKVEIK (SEQ YYCARVSPIAALMDYWGQGTLVT ID NO: 13) VSS (SEQ IDNO: 7) L1.GDF/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.NY/ASQSVGDFYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.APRLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQHSRDNSKNTLYLQMNSLRAEDTAV YNYPPSFTFGQGTKVEIK (SEQ YYCARVSPIAAPMDYWGQGTLVTID NO: 13) VSS (SEQ ID NO: 8) L1.GDF/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L3.NY/ ASQSVGDFYLAWYQQKPGQAPASGFTFGSYAMTWVRQAPGKGLE H3.AQ RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYNYPPSFTFGQGTKVEIK (SEQ YYCARVSPIAAQMDYWGQGTLVT ID NO: 14) VSS (SEQ IDNO: 3) L3.KW/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA P6E01ASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCKHYSRDNSKNTLYLQMNSLRAEDTAV GWPPSFTFGQGTKVEIK (SEQ IDYYCARVSPIASGMDYWGQGTLVT NO: 15) VSS (SEQ ID NO: 2) L3.PY/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA P6E01ASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVT ID NO: 16) VSS (SEQ IDNO: 2) L3.NY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA P6E01ASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYNYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVT ID NO: 17) VSS (SEQ IDNO: 2) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L1.PS/ASQSVSSSYPSWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE P6E01RLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVTID NO: 18) VSS (SEQ ID NO: 2) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.AH/ ASQSVSAHYLAWYQQKPGQAPASGFTFGSYAMTWVRQAPGKGLE P6E01 RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVT ID NO: 19) VSS (SEQ IDNO: 2) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L1.FF/ASQSVSSFFLAWYQQKPGQAPR ASGFTFGSYAMTWVRQAPGKGLE P6E01LLIYGASSRATGIPDRFSGSGSG WVSAISGSGGNTFYADSVKGRFTITDFTLTISRLEPEDFAVYYCQHYP SRDNSKNTLYLQMNSLRAEDTAV YPPSFTFGQGTKVEIK (SEQID YYCARVSPIASGMDYWGQGTLVT NO: 20) VSS (SEQ ID NO: 2) L3.PY/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L1.PH/ASQSVSPHYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE P6E01RLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVTID NO: 21) VSS (SEQ ID NO: 2) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L3.KY/ ASQSVSSSYLAWYQQKPGQAPASGFTFGSYAMTWVRQAPGKGLE P6E01 RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCKYYSRDNSKNTLYLQMNSLRAEDTAV PYPPSFTFGQGTKVEIK (SEQ IDYYCARVSPIASGMDYWGQGTLVT NO: 22) VSS (SEQ ID NO: 2) L3.PY/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.KF/ASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE P6E01RLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCKFY SRDNSKNTLYLQMNSLRAEDTAV PYPPSFTFGQGTKVEIK (SEQID YYCARVSPIASGMDYWGQGTLVT NO: 23) VSS (SEQ ID NO: 2) L3.PY/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA H2.QRASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADQRKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVT ID NO: 16) VSS (SEQ IDNO: 24) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA H2.DYASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE RLLIYGASSRATGIPDRFSGSGSWVSAIDYSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVT ID NO: 16) VSS (SEQ IDNO: 25) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA H2.YQASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE RLLIYGASSRATGIPDRFSGSGSWVSAISYQGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVT ID NO: 16) VSS (SEQ IDNO: 26) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA H2.LTASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE RLLIYGASSRATGIPDRFSGSGSWVSAISLTGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVT ID NO: 16) VSS (SEQ IDNO: 27) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA H2.HAASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE RLLIYGASSRATGIPDRFSGSGSWVSAISHAGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVT ID NO: 16) VSS (SEQ IDNO: 28) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA H2.QLASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADQLKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVT ID NO: 16) VSS (SEQ IDNO: 29) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA H3.YAASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIYAGMDYWGQGTLVT ID NO: 16) VSS (SEQ IDNO: 30) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA H3.AEASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIAAEMDYWGQGTLVT ID NO: 16) VSS (SEQ IDNO: 31) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA H3.AQASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIAAQMDYWGQGTLVT ID NO: 16) VSS (SEQ IDNO: 3) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA H3.TAQASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCTRVSPIAAQMDYWGQGTLVT ID NO: 16) VSS (SEQ IDNO: 32) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA P6E01ASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVT ID NO: 16) VSS (SEQ IDNO: 2) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L1.PS/ASQSVSSSYPSWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.QRRLLIYGASSRATGIPDRFSGSGS WVSAIVSGSGGNTFYADQRKGRFTIGTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQYYCARVSPIASGMDYWGQGTLVT ID NO: 18) VSS (SEQ ID NO: 24) L3.PY/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L1.PS/ASQSVSSSYPSWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.DYRLLIYGASSRATGIPDRFSGSGS WVSAIDYSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVTID NO: 18) VSS (SEQ ID NO: 25) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.PS/ ASQSVSSSYPSWYQQKPGQAPASGFTFGSYAMTWVRQAPGKGLE H2.YQ RLLIYGASSRATGIPDRFSGSGSWVSAISYQGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVT ID NO: 18) VSS (SEQ IDNO: 26) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L1.PS/ASQSVSSSYPSWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.LTRLLIYGASSRATGIPDRFSGSGS WVSAISLTGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVTID NO: 18) VSS (SEQ ID NO: 27) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.PS/ ASQSVSSSYPSWYQQKPGQAPASGFTFGSYAMTWVRQAPGKGLE H2.HA RLLIYGASSRATGIPDRFSGSGSWVSAISHAGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVT ID NO: 18) VSS (SEQ IDNO: 28) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L1.PS/ASQSVSSSYPSWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.QLRLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADQLKGRFTI GTDFTLTISRLEPEDFAVYYCQHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVTID NO: 18) VSS (SEQ ID NO: 29) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.PS/ ASQSVSSSYPSWYQQKPGQAPASGFTFGSYAMTWVRQAPGKGLE H3.YA RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIYAGMDYWGQGTLVT ID NO: 18) VSS (SEQ IDNO: 30) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L1.PS/ASQSVSSSYPSWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AERLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIAAEMDYWGQGTLVTID NO: 18) VSS (SEQ ID NO: 31) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.PS/ ASQSVSSSYPSWYQQKPGQAPASGFTFGSYAMTWVRQAPGKGLE H3.AQ RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIAAQMDYWGQGTLVT ID NO: 18) VSS (SEQ IDNO: 3) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L1.PS/ASQSVSSSYPSWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.TAQRLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCTRVSPIAAQMDYWGQGTLVTID NO: 18) VSS (SEQ ID NO: 32) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.AH/ ASQSVSAHYLAWYQQKPGQAPASGFTFGSYAMTWVRQAPGKGLE H2.QR RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADQRKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVT ID NO: 19) VSS (SEQ IDNO: 24) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L1.AH/ASQSVSAHYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.DYRLLIYGASSRATGIPDRFSGSGS WVSAIDYSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVTID NO: 19) VSS (SEQ ID NO: 25) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.AH/ ASQSVSAHYLAWYQQKPGQAPASGFTFGSYAMTWVRQAPGKGLE H2.YQ RLLIYGASSRATGIPDRFSGSGSWVSAISYQGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVT ID NO: 19) VSS (SEQ IDNO: 26) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L1.AH/ASQSVSAHYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.LTRLLIYGASSRATGIPDRFSGSGS WVSAISLTGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVTID NO: 19) VSS (SEQ ID NO: 27) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.AH/ ASQSVSAHYLAWYQQKPGQAPASGFTFGSYAMTWVRQAPGKGLE H2.HA RLLIYGASSRATGIPDRFSGSGSWVSAISHAGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVT ID NO: 19) VSS (SEQ IDNO: 28) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L1.AH/ASQSVSAHYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.QLRLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADQLKGRFTI GTDFTLTISRLEPEDFAVYYCQHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVTID NO: 19) VSS (SEQ ID NO: 29) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.AH/ ASQSVSAHYLAWYQQKPGQAPASGFTFGSYAMTWVRQAPGKGLE H3.YA RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIYAGMDYWGQGTLVT ID NO: 19) VSS (SEQ IDNO: 30) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L1.AH/ASQSVSAHYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AERLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIAAEMDYWGQGTLVTID NO: 19) VSS (SEQ ID NO: 31) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.AH/ ASQSVSAHYLAWYQQKPGQAPASGFTFGSYAMTWVRQAPGKGLE H3.AQ RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIAAQMDYWGQGTLVT ID NO: 19) VSS (SEQ IDNO: 3) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L1.AH/ASQSVSAHYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.TAQRLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCTRVSPIAAQMDYWGQGTLVTID NO: 19) VSS (SEQ ID NO: 32) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.FF/ ASQSVSSFFLAWYQQKPGQAPRASGFTFGSYAMTWVRQAPGKGLE H2.QR LLIYGASSRATGIPDRFSGSGSGWVSAISGSGGNTFYADQRKGRFTI TDFTLTISRLEPEDFAVYYCQHYPSRDNSKNTLYLQMNSLRAEDTAV YPPSFTFGQGTKVEIK (SEQ ID YYCARVSPIASGMDYWGQGTLVTNO: 20) VSS (SEQ ID NO: 24) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.FF/ ASQSVSSFFLAWYQQKPGQAPRASGFTFGSYAMTWVRQAPGKGLE H2.DY LLIYGASSRATGIPDRFSGSGSGWVSAIDYSGGNTFYADSVKGRFTI TDFTLTISRLEPEDFAVYYCQHYPSRDNSKNTLYLQMNSLRAEDTAV YPPSFTFGQGTKVEIK (SEQ ID YYCARVSPIASGMDYWGQGTLVTNO: 20) VSS (SEQ ID NO: 25) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.FF/ ASQSVSSFFLAWYQQKPGQAPRASGFTFGSYAMTWVRQAPGKGLE H2.YQ LLIYGASSRATGIPDRFSGSGSGWVSAISYQGGNTFYADSVKGRFTI TDFTLTISRLEPEDFAVYYCQHYPSRDNSKNTLYLQMNSLRAEDTAV YPPSFTFGQGTKVEIK (SEQ ID YYCARVSPIASGMDYWGQGTLVTNO: 20) VSS (SEQ ID NO: 26) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.FF/ ASQSVSSFFLAWYQQKPGQAPRASGFTFGSYAMTWVRQAPGKGLE H2.LT LLIYGASSRATGIPDRFSGSGSGWVSAISLTGGNTFYADSVKGRFTI TDFTLTISRLEPEDFAVYYCQHYPSRDNSKNTLYLQMNSLRAEDTAV YPPSFTFGQGTKVEIK (SEQ ID YYCARVSPIASGMDYWGQGTLVTNO: 20) VSS (SEQ ID NO: 27) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.FF/ ASQSVSSFFLAWYQQKPGQAPRASGFTFGSYAMTWVRQAPGKGLE H2.HA LLIYGASSRATGIPDRFSGSGSGWVSAISHAGGNTFYADSVKGRFTI TDFTLTISRLEPEDFAVYYCQHYPSRDNSKNTLYLQMNSLRAEDTAV YPPSFTFGQGTKVEIK (SEQ ID YYCARVSPIASGMDYWGQGTLVTNO: 20) VSS (SEQ ID NO: 28) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.FF/ ASQSVSSFFLAWYQQKPGQAPRASGFTFGSYAMTWVRQAPGKGLE H2.QL LLIYGASSRATGIPDRFSGSGSGWVSAISGSGGNTFYADQLKGRFTI TDFTLTISRLEPEDFAVYYCQHYPSRDNSKNTLYLQMNSLRAEDTAV YPPSFTFGQGTKVEIK (SEQ ID YYCARVSPIASGMDYWGQGTLVTNO: 20) VSS (SEQ ID NO: 29) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.FF/ ASQSVSSFFLAWYQQKPGQAPRASGFTFGSYAMTWVRQAPGKGLE H3.YA LLIYGASSRATGIPDRFSGSGSGWVSAISGSGGNTFYADSVKGRFTI TDFTLTISRLEPEDFAVYYCQHYPSRDNSKNTLYLQMNSLRAEDTAV YPPSFTFGQGTKVEIK (SEQ ID YYCARVSPIYAGMDYWGQGTLVTNO: 20) VSS (SEQ ID NO: 30) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.FF/ ASQSVSSFFLAWYQQKPGQAPRASGFTFGSYAMTWVRQAPGKGLE H3.AE LLIYGASSRATGIPDRFSGSGSGWVSAISGSGGNTFYADSVKGRFTI TDFTLTISRLEPEDFAVYYCQHYPSRDNSKNTLYLQMNSLRAEDTAV YPPSFTFGQGTKVEIK (SEQ ID YYCARVSPIAAEMDYWGQGTLVTNO: 20) VSS (SEQ ID NO: 31) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.FF/ ASQSVSSFFLAWYQQKPGQAPRASGFTFGSYAMTWVRQAPGKGLE H3.AQ LLIYGASSRATGIPDRFSGSGSGWVSAISGSGGNTFYADSVKGRFTI TDFTLTISRLEPEDFAVYYCQHYPSRDNSKNTLYLQMNSLRAEDTAV YPPSFTFGQGTKVEIK (SEQ ID YYCARVSPIAAQMDYWGQGTLVTNO: 20) VSS (SEQ ID NO: 3) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.FF/ ASQSVSSFFLAWYQQKPGQAPRASGFTFGSYAMTWVRQAPGKGLE H3.TAQ LLIYGASSRATGIPDRFSGSGSGWVSAISGSGGNTFYADSVKGRFTI TDFTLTISRLEPEDFAVYYCQHYPSRDNSKNTLYLQMNSLRAEDTAV YPPSFTFGQGTKVEIK (SEQ ID YYCTRVSPIAAQMDYWGQGTLVTNO: 20) VSS (SEQ ID NO: 32) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.PH/ ASQSVSPHYLAWYQQKPGQAPASGFTFGSYAMTWVRQAPGKGLE H2.QR RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADQRKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVT ID NO: 21) VSS (SEQ IDNO: 24) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L1.PH/ASQSVSPHYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.HARLLIYGASSRATGIPDRFSGSGS WVSAISHAGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIASGMDYWGQGTLVTID NO: 21) VSS (SEQ ID NO: 28) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.PH/ ASQSVSPHYLAWYQQKPGQAPASGFTFGSYAMTWVRQAPGKGLE H3.AE RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIAAEMDYWGQGTLVT ID NO: 21) VSS (SEQ IDNO: 31) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L1.PH/ASQSVSPHYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AQRLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCARVSPIAAQMDYWGQGTLVTID NO: 21) VSS (SEQ ID NO: 3) L3.PY/ EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA L1.PH/ ASQSVSPHYLAWYQQKPGQAPASGFTFGSYAMTWVRQAPGKGLE H3.TAQ RLLIYGASSRATGIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFTFGQGTKVEIK (SEQ YYCTRVSPIAAQMDYWGQGTLVT ID NO: 21) VSS (SEQ IDNO: 32) L3.PY/ EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.KY/ASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.QRRLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADQRKGRFTIGTDFTLTISRLEPEDFAVYYCKYY SRDNSKNTLYLQMNSLRAEDTAV PYPPSFTFGQGTKVEIK (SEQID YYCARVSPIASGMDYWGQGTLVT NO: 22) VSS (SEQ ID NO: 24) L3.PY/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.KY/ASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.DYRLLIYGASSRATGIPDRFSGSGS WVSAIDYSGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCKYY SRDNSKNTLYLQMNSLRAEDTAV PYPPSFTFGQGTKVEIK (SEQID YYCARVSPIASGMDYWGQGTLVT NO: 22) VSS (SEQ ID NO: 25) L3.PY/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.KY/ASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.YQRLLIYGASSRATGIPDRFSGSGS WVSAISYQGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCKYY SRDNSKNTLYLQMNSLRAEDTAV PYPPSFTFGQGTKVEIK (SEQID YYCARVSPIASGMDYWGQGTLVT NO: 22) VSS (SEQ ID NO: 26) L3.PY/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.KY/ASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.LTRLLIYGASSRATGIPDRFSGSGS WVSAISLTGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCKYY SRDNSKNTLYLQMNSLRAEDTAV PYPPSFTFGQGTKVEIK (SEQID YYCARVSPIASGMDYWGQGTLVT NO: 22) VSS (SEQ ID NO: 27) L3.PY/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.KY/ASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.HARLLIYGASSRATGIPDRFSGSGS WVSAISHAGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCKYY SRDNSKNTLYLQMNSLRAEDTAV PYPPSFTFGQGTKVEIK (SEQID YYCARVSPIASGMDYWGQGTLVT NO: 22) VSS (SEQ ID NO: 28) L3.PY/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.KY/ASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.QLRLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADQLKGRFTIGTDFTLTISRLEPEDFAVYYCKYY SRDNSKNTLYLQMNSLRAEDTAV PYPPSFTFGQGTKVEIK (SEQID YYCARVSPIASGMDYWGQGTLVT NO: 22) VSS (SEQ ID NO: 29) L3.PY/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.KY/ASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.YARLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCKYY SRDNSKNTLYLQMNSLRAEDTAV PYPPSFTFGQGTKVEIK (SEQID YYCARVSPIYAGMDYWGQGTLVT NO: 22) VSS (SEQ ID NO: 30) L3.PY/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.KY/ASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.TAQRLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCKYY SRDNSKNTLYLQMNSLRAEDTAV PYPPSFTFGQGTKVEIK (SEQID YYCTRVSPIAAQMDYWGQGTLVT NO: 22) VSS (SEQ ID NO: 32) L3.PY/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.KF/ASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.DYRLLIYGASSRATGIPDRFSGSGS WVSAIDYSGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCKFY SRDNSKNTLYLQMNSLRAEDTAV PYPPSFTFGQGTKVEIK (SEQID YYCARVSPIASGMDYWGQGTLVT NO: 23) VSS (SEQ ID NO: 25) L3.PY/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.KF/ASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.YQRLLIYGASSRATGIPDRFSGSGS WVSAISYQGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCKFY SRDNSKNTLYLQMNSLRAEDTAV PYPPSFTFGQGTKVEIK (SEQID YYCARVSPIASGMDYWGQGTLVT NO: 23) VSS (SEQ ID NO: 26) L3.PY/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.KF/ASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.LTRLLIYGASSRATGIPDRFSGSGS WVSAISLTGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCKFY SRDNSKNTLYLQMNSLRAEDTAV PYPPSFTFGQGTKVEIK (SEQID YYCARVSPIASGMDYWGQGTLVT NO: 23) VSS (SEQ ID NO: 27) L3.PY/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.KF/ASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.QLRLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADQLKGRFTIGTDFTLTISRLEPEDFAVYYCKFY SRDNSKNTLYLQMNSLRAEDTAV PYPPSFTFGQGTKVEIK (SEQID YYCARVSPIASGMDYWGQGTLVT NO: 23) VSS (SEQ ID NO: 29) L3.PY/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.KF/ASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.YARLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCKFY SRDNSKNTLYLQMNSLRAEDTAV PYPPSFTFGQGTKVEIK (SEQID YYCARVSPIYAGMDYWGQGTLVT NO: 23) VSS (SEQ ID NO: 30) L3.PY/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.KF/ASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AERLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCKFY SRDNSKNTLYLQMNSLRAEDTAV PYPPSFTFGQGTKVEIK (SEQID YYCARVSPIAAEMDYWGQGTLVT NO: 23) VSS (SEQ ID NO: 31) L3.PY/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.KF/ASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AQRLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCKFY SRDNSKNTLYLQMNSLRAEDTAV PYPPSFTFGQGTKVEIK (SEQID YYCARVSPIAAQMDYWGQGTLVT NO: 23) VSS (SEQ ID NO: 3) L3.PY/EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA L3.KF/ASQSVSSSYLAWYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.TAQRLLIYGASSRATGIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCKFY SRDNSKNTLYLQMNSLRAEDTAV PYPPSFTFGQGTKVEIK (SEQID YYCTRVSPIAAQMDYWGQGTLVT NO: 23) VSS (SEQ ID NO: 32) P5A2_VHVLEIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASQSVSSSYLAWYQQKPGQAPASGFTFSSYAMNWVRQAPGKGLE RLLMYDASIRATGIPDRFSGSGS WVSAISDSGGSTYYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCQQ SRDNSKNTLYLQMNSLRAEDTAV YGSWPLTFGQGTKVEIK (SEQID YYCARYWPMDIWGQGTLVTVSS NO: 34) (SEQ ID NO: 33) A02_Rd4_0.6nM_C06EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASQSVSVIYLAWYQQKPGQAPRASGFTFSSYAMNWVRQAPGKGLE LLMYDASIRATGIPDRFSGSGSG WVSAISDSGGSAWYADSVKGRFTTDFTLTISRLEPEDFAVYYCQQY ISRDNSKNTLYLQMNSLRAEDTAV QRWPLTFGQGTKVEIK (SEQID YYCARYWPMSLWGQGTLVTVSS NO: 36) (SEQ ID NO: 35) A02_Rd4_0.6nM_C09EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASQSVSSSYLAWYQQKPGQAPASGFTFSSYAMNWVRQAPGKGLE RLLMYDASIRATGIPDRFSGSGS WVSAISDSGGSMWYADSVKGRFGTDFTLTISRLEPEDFAVYYCQQ TISRDNSKNTLYLQMNSLRAEDTA YQSWPLTFGQGTKVEIK (SEQID VYYCARYWPMSLWGQGTLVTVS NO: 38) S (SEQ ID NO: 37) A02_Rd4_6nM_C16EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA (P5AC16)ASQSVSDIYLAWYQQKPGQAPR ASGFTFSSYAMNWVRQAPGKGLE LLMYDASIRATGIPDRFSGSGSGWVSAISdFGGSTYYADSVKGRFTI TDFTLTISRLEPEDFAVYYCQQY SRDNSKNTLYLQMNSLRAEDTAVQTWPLTFGQGTKVEIK (SEQ ID YYCARYWPMDIWGQGTLVTVSS NO: 40) (SEQ ID NO: 39)A02_Rd4_6nM_C03 EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCAASQSVSNLYLAWYQQKPGQAP ASGFTFSSYAMNWVRQAPGKGLE RLLMYDASIRATGIPDRFSGSGSWVSAISDSGGSTYYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQQ SRDNSKNTLYLQMNSLRAEDTAVYQGWPLTFGQGTKVEIK (SEQ ID YYCARYWPMDIWGQGTLVTVSS NO: 41) (SEQ ID NO: 33)A02_Rd4_6nM_C01 EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCAASQSVSAYYLAWYQQKPGQAP ASGFTFSSYAMNWVRQAPGKGLE RLLMYDASIRATGIPDRFSGSGSWVSAITASGGSTYYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQQ SRDNSKNTLYLQMNSLRAEDTAVYERWPLTFGQGTKVEIK (SEQ ID YYCARYWPMSLWGQGTLVTVSS NO: 43) (SEQ ID NO: 42)A02_Rd4_6nM_C26 EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCAASQSVSSLYLAWYQQKPGQAPR ASGFTFSSYAMNWVRQAPGKGLE LLMYDASIRATGIPDRFSGSGSGWVSAISDSGGSTYYADSVKGRFTI TDFTLTISRLEPEDFAVYYCQQY SRDNSKNTLYLQMNSLRAEDTAVQVWPLTFGQGTKVEIK (SEQ ID YYCARYWPMSLWGQGTLVTVSS NO: 45) (SEQ ID NO: 44)A02_Rd4_6nM_C25 EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCAASQSVSSSYLAWYQQKPGQAP ASGFTFSSYAMNWVRQAPGKGLE RLLMYDASIRATGIPDRFSGSGSWVSAISdSGGSRWYADSVKGRFT GTDFTLTISRLEPEDFAVYYCQQ ISRDNSKNTLYLQMNSLRAEDTAVYLDWPLTFGQGTKVEIK (SEQ ID YYCARYWPMTPWGQGTLVTVSS NO: 47) (SEQ ID NO: 46)A02_Rd4_6nM_C22 EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCAASQSVSSSYLAWYQQKPGQAP ASGFTFSSYAMNWVRQAPGKGLE RLLMYDASIRATGIPDRFSGSGSWVSAVLdSGGSTYYADSVKGRFT GTDFTLTISRLEPEDFAVYYCQQ ISRDNSKNTLYLQMNSLRAEDTAVYQVWPLTFGQGTKVEIK (SEQ ID YYCARYWPMTPWGQGTLVTVSS NO: 49) (SEQ ID NO: 48)A02_Rd4_6nM_C19 EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCAASQSVSVIYLAWYQQKPGQAPR ASGFTFSSYAMNWVRQAPGKGLE LLMYDASIRATGIPDRFSGSGSGWVSAISdSGGSRWYADSVKGRFT TDFTLTISRLEPEDFAVYYCQQYLISRDNSKNTLYLQMNSLRAEDTAV AWPLTFGQGTKVEIK (SEQ ID YYCARYWPMSDWGQGTLVTVSSNO: 51) (SEQ ID NO: 50) A02_Rd4_0.6nM_C03 EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA ASQSVSSSYLAWYQQKPGQAP ASGFTFSSYAMNWVRQAPGKGLERLLMYDASIRATGIPDRFSGSGS WVSAISdSGGSKWYADSVKGRFT GTDFTLTISRLEPEDFAVYYCQQISRDNSKNTLYLQMNSLRAEDTAV YFTWPLTFGQGTKVEIK (SEQ IDYYCARYWPMSLWGQGTLVTVSS NO: 53) (SEQ ID NO: 52) A02_Rd4_6nM_C07EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASQSVSPyYLAWYQQKPGQAPRASGFTFSSYAMNWVRQAPGKGLE LLMYDASIRATGIPDRFSGSGSG WVSAIGGSGGSLPYADSVKGRFTTDFTLTISRLEPEDFAVYYCQQYE ISRDNSKNTLYLQMNSLRAEDTAV RWPLTFGQGTKVEIK (SEQID YYCARYWPMDSWGQGTLVTVSS NO: 55) (SEQ ID NO: 54) A02_Rd4_6nM_C23EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASQSVSVEYLAWYQQKPGQAPASGFTFSSYAMNWVRQAPGKGLE RLLMYDASIRATGIPDRFSGSGS WVSAISdSGGSGWYADSVKGRFTGTDFTLTISRLEPEDFAVYYCQQ ISRDNSKNTLYLQMNSLRAEDTAV YARWPLTFGQGTKVEIK (SEQID YYCARYWPMSLWGQGTLVTVSS NO: 57) (SEQ ID NO: 56) A02_Rd4_0.6nM_C18EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASQSVSEIYLAWYQQKPGQAPRASGFTFSSYAMNWVRQAPGKGLE LLMYDASIRATGIPDRFSGSGSG WVSAVLdSGGSTYYADSVKGRFTTDFTLTISRLEPEDFAVYYCQQYF ISRDNSKNTLYLQMNSLRAEDTAV GWPLTFGQGTKVEIK (SEQID YYCARYWPMSLWGQGTLVTVSS NO: 59) (SEQ ID NO: 58) A02_Rd4_6nM_C10EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASQSVEMSYLAWYQQKPGQAPASGFTFSSYAMNWVRQAPGKGLE RLLMYDASIRATGIPDRFSGSGS WVSAISdSGGSCWYADSVKGRFTGTDFTLTISRLEPEDFAVYYCQQ ISRDNSKNTLYLQMNSLRAEDTAV YAHWPLTFGQGTKVEIK (SEQID YYCARYWPMTPWGQGTLVTVSS NO: 61) (SEQ ID NO: 60) A02_Rd4_6nM_C05EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASQSVSSSYLAWYQQKPGQAPASGFTFSSYAMNWVRQAPGKGLE RLLMYDASIRATGIPDRFSGSGS WVSAIFaSGGSTYYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCQQ SRDNSKNTLYLQMNSLRAEDTAV YQRWPLTFGQGTKVEIK (SEQID YYCARYWPMTPWGQGTLVTVSS NO: 63) (SEQ ID NO: 62) A02_Rd4_0.6nM_C10EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASQSVSAQYLAWYQQKPGQAPASGFTFSSYAMNWVRQAPGKGLE RLLMYDASIRATGIPDRFSGSGS WVSAISgWGGSLPYADSVKGRFTGTDFTLTISRLEPEDFAVYYCQQ ISRDNSKNTLYLQMNSLRAEDTAV YQRWPLTFGQGTKVEIK (SEQID YYCARYWPMDSWGQGTLVTVSS NO: 65) (SEQ ID NO: 64) A02_Rd4_6nM_C04EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASQSVSAIYLAWYQQKPGQAPRASGFTFSSYAMNWVRQAPGKGLE LLMYDASIRATGIPDRFSGSGSG WVSAIMsSGGPLYYADSVKGRFTITDFTLTISRLEPEDFAVYYCQQY SRDNSKNTLYLQMNSLRAEDTAV QVWPLTFGQGTKVEIK (SEQ IDYYCARYWPMALWGQGTLVTVSS NO: 67) (SEQ ID NO: 66) A02_Rd4_0.6nM_C26EIVLTQSPGTLSLSPGERATLSCG EVQLLESGGGLVQPGGSLRLSCA PSQSVSSSYLAWYQQKPGQAPRASGFTFSSYAMNWVRQAPGKGLE LLMYDASIRATGIPDRFSGSGSG WVSAILmSGGSTYYADSVKGRFTITDFTLTISRLEPEDFAVYYCQQY SRDNSKNTLYLQMNSLRAEDTAV QSWPLTFGQGTKVEIK (SEQ IDYYCARYWPMSLWGQGTLVTVSS NO: 69) (SEQ ID NO: 68) A02_Rd4_0.6nM_C13EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASQSVSSSYWAWYQQKPGQAPASGFTFSSYAMNWVRQAPGKGLE RLLMYDASIRATGIPDRFSGSGS WVSAISdSGGYRYYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCQQ SRDNSKNTLYLQMNSLRAEDTAV YESWPLTFGQGTKVEIK (SEQID YYCARYWPMSLWGQGTLVTVSS NO: 71) (SEQ ID NO: 70) A02_Rd4_0.6nM_C01EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA (P5AC1)GGQSVSSSYLAWYQQKPGQAP ASGFTFSSYAMNWVRQAPGKGLE RLLMYDASIRATGIPDRFSGSGSWVSAILsSGGSTYYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQQ SRDNSKNTLYLQMNSLRAEDTAVYQSWPLTFGQGTKVEIK (SEQ ID YYCARYWPMDIWGQGTLVTVSS NO: 73) (SEQ ID NO: 72)A02_Rd4_6nM_C08 EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCAASQSVSFIYLAWYQQKPGQAPR ASGFTFSSYAMNWVRQAPGKGLE LLMYDASIRATGIPDRFSGSGSGWVSAILdSGGSTYYADSVKGRFTI TDFTLTISRLEPEDFAVYYCQQY SRDNSKNTLYLQMNSLRAEDTAVGSWPLTFGQGTKVEIK (SEQ ID YYCARYWPMSPWGQGTLVTVSS NO: 75) (SEQ ID NO: 74)P5C1_VHVL EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA (PC1)ASQSVSSTYLAWYQQKPGQAPR ASGFTFSSYPMSWVRQAPGKGLE LLIYDASSRAPGIPDRFSGSGSGWVSAIGGSGGSTYYADSVKGRFT TDFTLTISRLEPEDFAVYYCQQYSISRDNSKNTLYLQMNSLRAEDTAV TSPLTFGQGTKVEIK (SEQ ID YYCARYWPMDSWGQGTLVTVSSNO: 77) (SEQ ID NO: 76) C01_Rd4_6nM_C24 EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA ASQSVSPEYLAWYQQKPGQAP ASGFTFSSYPMSWVRQAPGKGLERLLIYDASSRAPGIPDRFSGSGS WVSAIGGSGGSLPYADSVKGRFT GTDFTLTISRLEPEDFAVYYCQQISRDNSKNTLYLQMNSLRAEDTAV YSVWPLTFGQGTKVEIK (SEQ IDYYCARYWPMDSWGQGTLVTVSS NO: 79) (SEQ ID NO: 78) C01_Rd4_6nM_C26EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASQSVSAIYLAWYQQKPGQAPRASGFTFSSYPMSWVRQAPGKGLE LLIYDASSRAPGIPDRFSGSGSG WVSAIGGSGGSLPYADSVKGRFTTDFTLTISRLEPEDFAVYYCQQYS ISRDNSKNTLYLQMNSLRAEDTAV AWPLTFGQGTKVEIK (SEQID YYCARYWPMDSWGQGTLVTVSS NO: 317) (SEQ ID NO: 78) C01_Rd4_6nM_C10EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASQSVSSvYLAWYQQKPGQAPRASGFTFSSYPMSWVRQAPGKGLE LLIYDASSRAPGIPDRFSGSGSG WVSAIGgSGGSLPYADSVKGRFTITDFTLTISRLEPEDFAVYYCQQYS SRDNSKNTLYLQMNSLRAEDTAV TWPLTFGQGTKVEIK (SEQ IDYYCARYWPMDSWGQGTLVTVSS NO: 79) (SEQ ID NO: 78) C01_Rd4_0.6nM_C27EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASQSVSSTYLAWYQQKPGQAPRASGFTFSSYPMSWVRQAPGKGLE LLIYDASSRAPGIPDRFSGSGSG WVSAIGgSGGSLPYADSVKGRFTITDFTLTISRLEPEDFAVYYCQQYS SRDNSKNTLYLQMNSLRAEDTAV RWPLTFGQGTKVEIK (SEQ IDYYCARYWPMDSWGQGTLVTVSS NO: 81) (SEQ ID NO: 78) C01_Rd4_6nM_C20EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASQSVSPIYLAWYQQKPGQAPRASGFTFSSYPMSWVRQAPGKGLE LLIYDASSRAPGIPDRFSGSGSG WVSAIGgSGGSLPYADSVKGRFTITDFTLTISRLEPEDFAVYYCQQYS SRDNSKNTLYLQMNSLRAEDTAV AFPLTFGQGTKVEIK (SEQ IDYYCARYWPMDSWGQGTLVTVSS NO: 82) (SEQ ID NO: 78) C01_Rd4_6nM_C12EIVLTQSPGTLSLSPGERATLSC EVQLLESGGGLVQPGGSLRLSCA (PC1C12)WLSQSVSSTYLAWYQQKPGQA ASGFTFSSYPMSWVRQAPGKGLE PRLLIYDASSRAPGIPDRFSGSGWVSAIGgSGGWSYYADSVKGRFT SGTDFTLTISRLEPEDFAVYYCQ ISRDNSKNTLYLQMNSLRAEDTAVQYSEWPLTFGQGTKVEIK (SEQ YYCARYWPMDSWGQGTLVTVSS ID NO: 84) (SEQ ID NO:83) C01_Rd4_0.6nM_C16 EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCAASQSVSSTYLAWYQQKPGQAPR ASGFTFSSYPMSWVRQAPGKGLE LLIYDASSRAPGIPDRFSGSGSGWVSAIGgSGGSLPYADSVKGRFTI TDFTLTISRLEPEDFAVYYCQQYSSRDNSKNTLYLQMNSLRAEDTAV SWPLTFGQGTKVEIK (SEQ ID YYCARYWPMDSWGQGTLVTVSSNO: 85) (SEQ ID NO: 78) C01_Rd4_0.6nM_C09 EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA ASQSVSSIFLAWYQQKPGQAPR ASGFTFSSYPMSWVRQAPGKGLELLIYDASSRAPGIPDRFSGSGSG WVSAIGgSGGSLPYADSVKGRFTITDFTLTISRLEPEDFAVYYCQQYS SRDNSKNTLYLQMNSLRAEDTAV AWPLTFGQGTKVEIK (SEQ IDYYCARYWPMDSWGQGTLVTVSS NO: 86) (SEQ ID NO: 78) C01_Rd4_6nM_C09EIVLTQSPGTLSLSPGERATLSCA EVQLLESGGGLVQPGGSLRLSCA CSQSVSSTYLAWYQQKPGQAPRASGFTFSSYPMSWVRQAPGKGLE LLIYDASSRAPGIPDRFSGSGSG WVSATVgSGGSIGYADSVKGRFTITDFTLTISRLEPEDFAVYYCQQYS SRDNSKNTLYLQMNSLRAEDTAV AWPLTFGQGTKVEIK (SEQ IDYYCARYWPMDSWGQGTLVTVSS NO: 88) (SEQ ID NO: 87) C01_Rd4_0.6nM_C03EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASCDVSSTYLAWYQQKPGQAPRASGFTFSSYPMSWVRQAPGKGLE LLIYDASSRAPGIPDRFSGSGSG WVSAIGgSGGSLPYADSVKGRFTITDFTLTISRLEPEDFAVYYCQQY SRDNSKNTLYLQMNSLRAEDTAV MRSPLTFGQGTKVEIK (SEQ IDYYCARYWPMDSWGQGTLVTVSS NO: 89) (SEQ ID NO: 78) C01_Rd4_0.6nM_C06EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASEAVPSTYLAWYQQKPGQAPRASGFTFSSYPMSWVRQAPGKGLE LLIYDASSRAPGIPDRFSGSGSG WVSAIGgSGGSLPYADSVKGTISRTDFTLTISRLEPEDFAVYYCQQYS DNSKNTLYLQMNSLRAEDTAVYY AFPLTFGQGTKVEIK (SEQ IDCARYWPMDSWGQGTLVTVSS NO: 90) (SEQ ID NO: 78) C01_Rd4_6nM_C04EIVLTQSPGTLSLSPGERATLSCC EVQLLESGGGLVQPGGSLRLSCA SSQSVSSTYLAWYQQKPGQAPRASGFTFSSYPMSWVRQAPGKGLE LLIYDASSRAPGIPDRFSGSGSG WVSAIGgSGGSLPYADSVKGRFTITDFTLTISRLEPEDFAVYYCQQYS SRDNSKNTLYLQMNSLRAEDTAV AFPLTFGQGTKVEIK (SEQ IDYYCARYWPMDSWGQGTLVTVSS NO: 91) (SEQ ID NO: 78) COMBO_Rd4_0.6nM_C22EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA (COM22)ASVRVSSTYLAWYQQKPGQAPR ASGFTFSSYAMNWVRQAPGKGLE LLMYDASIRATGIPDRFSGSGSGWVSAISdSGGSRWYADSVKGRFT TDFTLTISRLEPEDFAVYYCQQY ISRDNSKNTLYLQMNSLRAEDTAVMKWPLTFGQGTKVEIK (SEQ ID YYCTRYWPMDIWGQGTLVTVSS NO: 93) (SEQ ID NO: 92)COMBO_Rd4_6nM_C21 EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCAASQSVSAAYLAWYQQKPGQAP ASGFTFSSYPMSWVRQAPGKGLE RLLMYDASIRATGIPDRFSGSGSWVSAIGgSGGSLPYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQQ SRDNSKNTLYLQMNSLRAEDTAVYMCWPLTFGQGTKVEIK (SEQ ID YYCARYWPMDSWGQGTLVTVSS NO: 94) (SEQ ID NO: 78)COMBO_Rd4_6nM_C10 EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCAASQSVSSSYWGWYQQKPGQAP ASGFTFSSYPMSWVRQAPGKGLE RLLMYDASIRATGIPDRFSGSGSWVSAIGgSGGSIHYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQQ SRDNSKNTLYLQMNSLRAEDTAVYQCWPLTFGQGTKVEIK (SEQ ID YYCARYWPMDSWGQGTLVTVSS NO: 96) (SEQ ID NO: 95)COMBO_Rd4_0.6nM_C04 EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCAASQSVSSTYLAWYQQKPGQAPR ASGFTFSSYPMSWVRQAPGKGLE LLMYDASIRATGIPDRFSGSGSGWVSAHIgSGGSTYYADSVKGRFTI TDFTLTISRLEPEDFAVYYCQQY SRDNSKNTLYLQMNSLRAEDTAVQSWPLTFGQGTKVEIK (SEQ ID YYCARYWPMDSWGQGTLVTVSS NO: 98) (SEQ ID NO: 97)COMBO_Rd4_6nM_C25 EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCAASQSVSSpYLAWYQQKPGQAPR ASGFTFSSYPMSWVRQAPGKGLE LLMYDASIRATGIPDRFSGSGSGWVSAIGgSGGSTYYADSVKGRFTI TDFTLTISRLEPEDFAVYYCQQY SRDNSKNTLYLQMNSLRAEDTAVQSWPLTFGQGTKVEIK (SEQ ID YYCARYWPMDPWGQGTLVTVSS NO: 100) (SEQ ID NO: 99)COMBO_Rd4_0.6nM_C21 EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCAASQSVSSSYLAWYQQKPGQAP ASGFTFSSYPMSWVRQAPGKGLE RLLMYDASIRATGIPDRFSGSGSWVSAIGgSGGSLPYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQQ SRDNSKNTLYLQMNSLRAEDTAVYQSWPLTFGQGTKVEIK (SEQ ID YYCARYWPMDSWGQGTLVTVSS NO: 38) (SEQ ID NO: 78)COMBO_Rd4_6nM_C11 EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCAASQSVSPIYLAWYQQKPGQAPR ASGFTFSSYPMSWVRQAPGKGLE LLMYDASIRATGIPDRFSGSGSGWVSAIGGSGGSLGYADSVKGRFT TDFTLTISRLEPEDFAVYYCQQY ISRDNSKNTLYLQMNSLRAEDTAVKAWPLTFGQGTKVEIK (SEQ ID YYCARYWPMDSWGQGTLVTVSS NO: 102) (SEQ ID NO:101) COMBO_Rd4_0.6nM_C20 EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA ASQSVSYLYLAWYQQKPGQAPR ASGFTFSSYPMSWVRQAPGKGLELLMYDASIRATGIPDRFSGSGSG WVSAIGGSGGSLPYADSVKGRFT TDFTLTISRLEPEDFAVYYCQQYISRDNSKNTLYLQMNSLRAEDTAV MEWPLTFGQGTKVEIK (SEQ ID YYCARYWPMDSWGQGTLVTVSSNO: 103) (SEQ ID NO: 78) COMBO_Rd4_6nM_C09 EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA ASQSVSAQYLAWYQQKPGQAP ASGFTFSSYPMSWVRQAPGKGLERLLMYDASIRATGIPDRFSGSGS WVSAIFASGGSTYYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQQSRDNSKNTLYLQMNSLRAEDTAV YQAWPLTFGQGTKVEIK (SEQ ID YYCARYWPMDSWGQGTLVTVSSNO: 105) (SEQ ID NO: 104) COMBO_Rd4_6nM_C08 EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA ASQSVSSSYLAWYQQKPGQAP ASGFTFSSYPMSWVRQAPGKGLERLLMYDASIRATGIPDRFSGSGS WVSAIGGSGTWTYYADSVKGRFT GTDFTLTISRLEPEDFAVYYCQQISRDNSKNTLYLQMNSLRAEDTAV YQKWPLTFGQGTKVEIK (SEQ IDYYCARYWPMDSWGQGTLVTVSS NO: 107) (SEQ ID NO: 106) COMBO_Rd4_0.6nM_C19EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASQSVSAVYLAWYQQKPGQAPASGFTFSSYPMSWVRQAPGKGLE RLLMYDASIRATGIPDRFSGSGS WVSAIGGSGGSLPYADSVKGRFTGTDFTLTISRLEPEDFAVYYCQQ ISRDNSKNTLYLQMNSLRAEDTAV YRAWPLTFGQGTKVEIK (SEQID YYCARYWPMDSWGQGTLVTVSS NO: 108) (SEQ ID NO: 78) COMBO_Rd4_0.6nM_C02EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASIAVSSTYLAWYQQKPGQAPRASGFTFSSYPMSWVRQAPGKGLE LLMYDASIRATGIPDRFSGSGSG WVSAIGGSGGSLPYADSVKGRFTTDFTLTISRLEPEDFAVYYCQQY ISRDNSKNTLYLQMNSLRAEDTAV MVWPLTFGQGTKVEIK (SEQID YYCARYWPMDSWGQGTLVTVSS NO: 109) (SEQ ID NO: 78) COMBO_Rd4_0.6nM_C23EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA PRQSVSSSYLAWYQQKPGQAPASGFTFSSYPMSWVRQAPGKGLE RLLMYDASIRATGIPDRFSGSGS WVSALFGSGGSTYYADSVKGRFTGTDFTLTISRLEPEDFAVYYCQQ ISRDNSKNTLYLQMNSLRAEDTAV YQDWPLTFGQGTKVEIK (SEQID YYCARYWPMDSWGQGTLVTVSS NO: 111) (SEQ ID NO: 110) COMBO_Rd4_0.6nM_C29EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASQSVSSSYLAWYQQKPGQAPASGFTFSSYPMSWVRQAPGKGLE RLLMYDASIRATGIPDRFSGSGS WVSAIGGSGGSLPYADSVKGRFTGTDFTLTISRLEPEDFAVYYCQQ ISRDNSKNTLYLQMNSLRAEDTAV YQSWPLTFGQGTKVEIK (SEQID YYCARYWPMDIWGQGTLVTVSS NO: 38) (SEQ ID NO: 112) COMBO_Rd4_0.6nM_C09EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASQSVSSTYLAWYQQKPGQAPRASGFTFSSYPMSWVRQAPGKGLE LLMYDASIRATGIPDRFSGSGSG WVSAIGGSGGSLPYADSVKGRFTTDFTLTISRLEPEDFAVYYCQQY ISRDNSKNTLYLQMNSLRAEDTAV QEWPLTFGQGTKVEIK (SEQID YYCARYWPMDIWGQGTLVTVSS NO: 113) (SEQ ID NO: 112) COMBO_Rd4_6nM_C12EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASQSVSASYLAWYQQKPGQAPASGFTFSSYPMSWVRQAPGKGLE RLLMYDASIRATGIPDRFSGSGS WVSAALGSGGSTYYADSVKGRFGTDFTLTISRLEPEDFAVYYCQQ TISRDNSKNTLYLQMNSLRAEDTA YMSWPLTFGQGTKVEIK (SEQID VYYCARYWPMDSWGQGTLVTVS NO: 115) S (SEQ ID NO: 114)COMBO_Rd4_0.6nM_C30 EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCAASQSVSYMYLAWYQQKPGQAP ASGFTFSSYPMSWVRQAPGKGLE RLLIYDASIRATGIPDRFSGSGSGWVSAIGGSGGSTYYADSVKGRFT TDFTLTISRLEPEDFAVYYCQQY ISRDNSKNTLYLQMNSLRAEDTAVKSWPLTFGQGTKVEIK (SEQ ID YYCARYWPMDSWGQGTLVTVSS NO: 116) (SEQ ID NO: 76)COMBO_Rd4_0.6nM_C14 EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCAASQSVSALYLAWYQQKPGQAP ASGFTFSSYPMSWVRQAPGKGLE RLLMYDASIRATGIPDRFSGSGSWVSAIGGSGGSLPYADSVKGRFT GTDFTLTISRLEPEDFAVYYCQQ ISRDNSKNTLYLQMNSLRAEDTAVYYGWPLTFGQGTKVEIK (SEQ ID YYCARYWPMDIWGQGTLVTVSS NO: 117) (SEQ ID NO:112) COMBO_Rd4_6nM_C07 EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCAASQPISSSYLAWYQQKPGQAPR ASGFTFSSYPMSWVRQAPGKGLE LLMYDASIRATGIPDRFSGSGSGWVSAIGGSGGSLPYADSVKGRFT TDFTLTISRLEPEDFAVYYCQQY ISRDNSKNTLYLQMNSLRAEDTAVQGWPLTFGQGTKVEIK (SEQ ID YYCARYWPMADWGQGTLVTVSS NO: 119) (SEQ ID NO:118) COMBO_Rd4_6nM_C02 EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCAASQSVSSSYLAWYQQKPGQAP ASGFTFSSYAMNWVRQAPGKGLE RLLMYDASIRATGIPDRFSGSGSWVSAISDSGGFVYYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQQ SRDNSKNTLYLQMNSLRAEDTAVYEFWPLTFGQGTKVEIK (SEQ ID YYCARYWPMDSWGQGTLVTVSS NO: 121) (SEQ ID NO:120) COMBO_Rd4_0.6nM_C05 EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA ASQSVSSTYLAWYQQKPGQAPR ASGFTFSSYAMNWVRQAPGKGLELLMYDASIRATGIPDRFSGSGSG WVSAIGGSGGSTYYADSVKGRFT TDFTLTISRLEPEDFAVYYCQQYISRDNSKNTLYLQMNSLRAEDTAV MSWPLTFGQGTKVEIK (SEQ ID YYCARYWPMSLWGQGTLVTVSSNO: 123) (SEQ ID NO: 122) COMBO_Rd4_0.6nM_C17 EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA ASQGISSTYLAWYQQKPGQAPR ASGFTFSSYPMSWVRQAPGKGLELLMYDASIRATGIPDRFSGSGSG WVSAIGGSGGSLPYADSVKGRFT TDFTLTISRLEPEDFAVYYCQQYISRDNSKNTLYLQMNSLRAEDTAV AYWPLTFGQGTKVEIK (SEQ ID YYCARYWPMDIWGQGTLVTVSSNO: 124) (SEQ ID NO: 112) COMBO_Rd4_6nM_C22 EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA ASQSVSSSYLAWYQQKPGQAP ASGFTFSSYAMNWVRQAPGKGLERLLMYDASIRATGIPDRFSGSGS WVSACLDSGGSTYYADSVKGRFT GTDFTLTISRLEPEDFAVYYCQQISRDNSKNTLYLQMNSLRAEDTAV YQGWPLTFGQGTKVEIK (SEQ IDYYCARYWPMDSWGQGTLVTVSS NO: 126) (SEQ ID NO: 125) COMBO_Rd4_0.6nM_C11EIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASQSVSVRYLAWYQQKPGQAPASGFTFSSYPMSWVRQAPGKGLE RLLMYDASIRATGIPDRFSGSGS WVSAALGSGGSTYYADSVKGRFGTDFTLTISRLEPEDFAVYYCQQ TISRDNSKNTLYLQMNSLRAEDTA YGSWPITFGQGTKVEIK (SEQID VYYCARYWPMSLWGQGTLVTVS NO: 128) S (SEQ ID NO: 127) P6DYEIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASQSVSSSYPSWYQQKPGQAPASGFTFGSYAMTWVRQAPGKGLE RLLIYGASSRATGIPDRFSGSGS WVSAIDYSGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQYYCARVSPIASGMDYWGQGTLVT ID NO: 18) VSS (SEQ ID NO: 25) P6APEIVLTQSPGTLSLSPGERATLSCR EVQLLESGGGLVQPGGSLRLSCA ASQLGSFYLAWYQQKPGQAPRLASGFTFGSYAMTWVRQAPGKGLE LIYGASSRATGIPDRFSGSGSGT WVSAISGSGGNTFYADSVKGRFTIDFTLTISRLEPEDFAVYYCQHYN SRDNSKNTLYLQMNSLRAEDTAV YPPSFTFGQGTKVEIK (SEQ IDYYCARVSPIAAPMDYWGQGTLVT NO: 80) VSS (SEQ ID NO: 8) ConsensusEIVLTQSPGTLSLSPGERATLSC EVQLLESGGGLVQPGGSLRLSCAX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂WY ASGFTFX₁SYX₂MX₃WVRQAPGKGQQKPGQAPRLLMYX₁₃ASX₁₄RAX₁₅ LEWVSAX₄X₅X₆X₇GX₈X₉X₁₀X₁₁YADGIPDRFSGSGSGTDFTLTISRLE X₁₂X₁₃KGRFTISRDNSKNTLYLQMNPEDFAVYYCX₁₆X₁₇YX₁₈X₁₉PPSF SLRAEDTAVYYCARVSPIX₁₄X₁₅X₁₆ TFGQGTKVEIK,wherein X₁ is R, MDYWGQGTLVTVSS, wherein X₁ G, W, A, or C; X₂ is A, P,G, L, C, is G or S, X₂ is A or P; X₃ is T, N, or or S; X₃ is S, G, or R;X₄ is Q, C, S; X₄ is I, V, T, H, L, A, or C; X₅ is S, E, V, or I; X₅ isS, P, G, A, R, or D; D, G, T, I, L, F, M, or V; X₆ is G, Y, X₆ is V, G,I, or L; X₇ is S, E, D, P, L, H, D, A, S, or M; X₇ is S, Q, T, A, or G;X₈ is S, P, F, A, M, E, V, N, F, or W; X₈ is G or T; X₉ is N, S, P, D,or Y; X₉ is I, T, V, E, S, A, M, Q, Y, W, or F; X₁₀ is S, T, I, L, T, A,R, Y, H, R, or F; X₁₀ is Y or F; X₁₁ is V, K, G, or C; X₁₁ is F, Y, P,W, H, L, W, or P; X₁₂ is A, S, or G, X₁₃ is or G; X₁₂ is V, R, or L; X₁₃is G or T; G or D; X₁₄ is S or I; X₁₅ is T or P; X₁₄ is A or Y; X₁₅ is Aor S; and X₁₆ is X₁₆ is Q or K; X₁₇ is H or Y; X₁₈ is G, Q, L, P, or E(SEQ ID NO: 313); G, N, or P; and X₁₉ is S, W, or Y or (SEQ ID NO: 315);or EVQLLESGGGLVQPGGSLRLSCA EIVLTQSPGTLSLSPGERATLSCASGFTFX₁SYX₂MX₃WVRQAPGKG X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂WYLEWVSAX₄X₅X₆X₇GX₈X₉X₁₀X₁₁YAD QQKPGQAPRLLMYX₁₃ASX₁₄RAX₁₅X₁₂X₁₃KGRFTISRDNSKNTLYLQMN GIPDRFSGSGSGTDFTLTISRLESLRAEDTAVYYCARYWPMX₁₄X₁₅ PEDFAVYYCQQYX₁₆X₁₇X₁₈PX₁₉F WGQGTLVTVSS, whereinX₁ is G GQGTKVEIK, wherein X₁ is R, G, or S, X₂ is A or P; X₃ is T, N,or S; W, A, or C; X₂ is A, P, G, L, C, or X₄ is I, V, T, H, L, A, or C;X₅ is S, D, S; X₃ is S, G, or R; X₄ is Q, C, E, G, T, I, L, F, M, or V;X₆ is G, Y, L, V, or I; X₅ is S, L, P, G, A, R, or D; H, D, A, S, or M;X₇ is S, Q, T, A, F, X₆ is V, G, or I; X₇ is S, E, D, or P; or W; X₈ isG or T; X₉ is N, S, P, Y, X₈ is S, P, F, A, M, E, V, N, D, or W, or F;X₁₀ is S, T, I, L, T, A, R, V, Y; X₉ is I, T, V, E, S, A, M, Q, Y, H, K,G, or C; X₁₁ is F, Y, P, W, H, or or R; X₁₀ is Y or F; X₁₁ is L, W, orG; X₁₂ is V, R, or L; X₁₃ is G or T; P; X₁₂ is A, S, or G, X₁₃ is G orD; X₁₄ is D, S, T, or A; and X₁₅ is I, S, L, X₁₄ is S or I; X₁₅ is T orP; X₁₆ is G, P, or D (SEQ ID NO: 314) Q, E, L, F, A, S, M, R, K, or Y;X₁₇ is S, R, T, G, R, V, D, A, H, E, K, C, F, or Y; X₁₈ is W, S, or F;and X₁₉ is L or I (SEQ ID NO: 316) P4G4 EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA ASQSVSSSYLAWYQQKPGQAP ASGFTFSSYAMSWVRQAPGKGLERLLIYGASSRAYGIPDRFSGSGS WVSAISASGGSTYYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQHSRDNSKNTLYLQMNSLRAEDTAV YGSPPLFTFGQGTKVEIK (SEQ YYCARLSWSGAFDNWGQGTLVTID NO: 401) VSS (SEQ ID NO: 378) P1A11 EIVLTQSPGTLSLSPGERATLSCREVQLLESGGGLVQPGGSLRLSCA ASQNVSSSYLAWYQQKPGQAP ASGFTFRSYAMSWVRQAPGKGLERLLIYGASYRATGIPDRFSGSGS WVSAISGSGGSTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQHSRDNSKNTLYLQMNSLRAEDTAV YGSPPSFTFGQGTKVEIK (SEQ YYCATVGTSGAFGIWGQGTLVTVID NO: 379) SS (SEQ ID NO: 380)

Also provided herein are CDR portions of extracellular ligand-bindingdomains of CARs to BCMA (including Chothia, Kabat CDRs, and CDR contactregions). Determination of CDR regions is well within the skill of theart. It is understood that in some embodiments, CDRs can be acombination of the Kabat and Chothia CDR (also termed “combined CRs” or“extended CDRs”). In some embodiments, the CDRs are the Kabat CDRs. Inother embodiments, the CDRs are the Chothia CDRs. In other words, inembodiments with more than one CDR, the CDRs may be any of Kabat,Chothia, combination CDRs, or combinations thereof. Table 2 providesexamples of CDR sequences provided herein.

TABLE 2 Heavy Chain mAb CDRH1 CDRH2 CDRH3 P6E01 SYAMT (SEQ ID NO:AISGSGGNTFYA VSPIASGMD For the following mAbs: 129) (Kabat); DSVKG (SEQID Y (SEQ ID P6E01/P6E01; L1.LGF/L3.KW/ GFTFGSY (SEQ ID NO: 132) (Kabat)NO: 134) P6EO1; NO: 130) (Chothia); SGSGGN (SEQ L1.LGF/L3.NY/P6E01;GFTFGSYAMT (SEQ ID NO: 133) L1.GDF/L3.NY/P6E01; ID NO: 131) (Chothia)L3.KW/P6E01; (extended) L3.PY/P6E01; L3.NY/P6E01; L3.PY/L1.PS/P6E01;L3.PY/L1.AH/P6E01; L3.PY/L1.FF/P6E01; L3.PY/L1.PH/P6E01;L3.PY/L3.KY/P6E01; L3.PY/L3.KF/P6E01; and L3.PY/P6E01. H3.AQ SYAMT (SEQID NO: AISGSGGNTFYA VSPIAAQMD For the following mAbs: 129) (Kabat);DSVKG (SEQ ID Y (SEQ ID P6E01/H3.AQ; GFTFGSY (SEQ ID NO: 132) (Kabat)NO: 135) L1.LGF/L3.KW/H3.AQ; NO: 130) (Chothia); SGSGGN (SEQL1.LGF/L3.PY/H3.AQ GFTFGSYAMT (SEQ ID NO: 133) ID NO: 131) (Chothia)(extended) H3.AL SYAMT (SEQ ID NO: AISGSGGNTFYA VSPIAALMDY For thefollowing mAbs: 129) (Kabat); DSVKG (SEQ ID (SEQ ID NO:L1.LGF/L3.KW/H3.AL; GFTFGSY (SEQ ID NO: 132) (Kabat) 136)L1.LGF/L3.NY/H3.AL; NO: 130) (Chothia); SGSGGN (SEQ and GFTFGSYAMT (SEQID NO: 133) L1.GDF/L3.NY/H3.AL. ID NO: 131) (Chothia) (extended) H3.APSYAMT (SEQ ID NO: AISGSGGNTFYA VSPIAAPMDY For the following mAbs: 129)(Kabat); DSVKG (SEQ ID (SEQ ID NO: L1.LGF/L3.KW/H3.AP; GFTFGSY (SEQ IDNO: 132) (Kabat) 137) L1.LGF/L3.PY/H3.AP; NO: 130) (Chothia); SGSGGN(SEQ L1.LGF/L3NY/H3.AP; GFTFGSYAMT (SEQ ID NO: 133) L1.GDF/L3.KW/H3.AP;ID NO: 131) (Chothia) L1.GDF/L3NY/H3.AP; (extended) P6AP. H2.QR SYAMT(SEQ ID NO: AISGSGGNTFYA VSPIASGMD For the following mAbs: 129) (Kabat);DQRKG (SEQ ID Y (SEQ ID L3.PY/H2.QR; GFTFGSY (SEQ ID NO: 138) (Kabat)NO: 134) L3.PY/L1.PS/H2.QR; NO: 130) (Chothia); SGSGGN (SEQL3.PY/L1.AH/H2.QR; GFTFGSYAMT (SEQ ID NO: 133) L3.PY/L1.FF/H2.QR; ID NO:131) (Chothia) L3.PY/L1.PH/H2.QR; (extended) and L3.PY/L3.KY/H2.QR.H2.DY SYAMT (SEQ ID NO: AIDYSGGNTFYA VSPIASGMD For the following mAbs:129) (Kabat); DSVKG (SEQ ID Y (SEQ ID L3.PY/H2.DY; P6DY; GFTFGSY (SEQ IDNO: 139) (Kabat) NO: 134) L3.PY/L1.PS/H2.DY; NO: 130) (Chothia); DYSSGN(SEQ L3.PY/L1.AH/H2.DY; GFTFGSYAMT (SEQ ID NO: 140) L3.PY/L1.FF/H2.DY;ID NO: 131) (Chothia) L3.PY/L3.KY/H2.DY; (extended) andL3.PY/L3.KF/H2.DY. H2.YQ SYAMT (SEQ ID NO: AISYQGGNTFYA VSPIASGMD Forthe following mAbs: 129) (Kabat); DSVKG (SEQ ID Y (SEQ ID L3.PY/H2.YQ;GFTFGSY (SEQ ID NO: 141) (Kabat) NO: 134) L3.PY/L1.PS/H2.YQ; NO: 130)(Chothia); SYQGGN (SEQ L3.PY/L1.AH/H2.YQ; GFTFGSYAMT (SEQ ID NO: 142)L3.PY/L1.FF/H2.YQ; ID NO: 131) (Chothia) L3.PY/L3.KY/H2.YQ; (extended)and L3.PY/L3.KF/H2.YQ. H2.LT SYAMT (SEQ ID NO: AISLTGGNTFYA VSPIASGMDFor the following mAbs: 129) (Kabat); DSVKG (SEQ ID Y (SEQ IDL3.PY/H2.LT; GFTFGSY (SEQ ID NO: 143) (Kabat) NO: 134)L3.PY/L1.PS/H2.LT; NO: 130) (Chothia); SLTGGN (SEQ L3.PY/L1.AH/H2.LT;GFTFGSYAMT (SEQ ID NO: 144) L3.PY/L1.FF/H2.LT; ID NO: 131) (Chothia)L3.PY/L3.KY/H2.LT; (extended) and L3.PY/L3.KF/H2.LT. H2.HA SYAMT (SEQ IDNO: AISHAGGNTFYA VSPIASGMD For the following mAbs: 129) (Kabat); DSVKG(SEQ ID Y (SEQ ID L3.PY/H2.HA; GFTFGSY (SEQ ID NO: 145) (Kabat) NO: 134)L3.PY/L1.AH/H2.HA; NO: 130) (Chothia); SHAGGN (SEQ L3.PY/L1.FF/H2.HA;GFTFGSYAMT (SEQ ID NO: 146) L3.PY/L1.PH/H2.HA; ID NO: 131) (Chothia) and(extended) L3.PY/L3.KY/H2.HA. H2.QL SYAMT (SEQ ID NO: AISGSGGNTFYAVSPIASGMD For the following mAbs: 129) (Kabat); DQLKG (SEQ ID Y (SEQ IDL3.PY/H2.QL; GFTFGSY (SEQ ID NO: 147) (Kabat) NO: 134)L3.PY/L1.PS/H2.QL; NO: 130) (Chothia); SGSGGN (SEQ L3.PY/L1.AH/H2.QL;GFTFGSYAMT (SEQ ID NO: 133) L3.PY/L1.FF/H2.QL; ID NO: 131) (Chothia)L3.PY/L3.KY/H2.QL; (extended) and L3.PY/L3.KF/H2.QL. H3.YA SYAMT (SEQ IDNO: AISGSGGNTFYA VSPIYAGMD For the following mAbs: 129) (Kabat); DSVKG(SEQ ID Y (SEQ ID L3.PY/H3.YA; GFTFGSY (SEQ ID NO: 132) (Kabat) NO: 148)L3.PY/L1.PS/H3.YA; NO: 130) (Chothia); SGSGGN (SEQ L3.PY/L1.AH/H3.YA;GFTFGSYAMT (SEQ ID NO: 133) L3.PY/L1.FF/H3.YA; ID NO: 131) (Chothia)L3.PY/L3.KY/H3.YA; (extended) and L3.PY/L3.KF/H3.YA. H3.AE SYAMT (SEQ IDNO: AISGSGGNTFYA VSPIAAEMD For the following mAbs: 129) (Kabat); DSVKG(SEQ ID Y (SEQ ID L3.PY/H3.AE; GFTFGSY (SEQ ID NO: 132) (Kabat) NO: 149)L3.PY/L1.AH/H3.AE; NO: 130) (Chothia); SGSGGN (SEQ L3.PY/L1.FF/H3.AE;GFTFGSYAMT (SEQ ID NO: 133) L3.PY/L1.PH/H3.AE; ID NO: 131) (Chothia) and(extended) L3.PY/L3.KF/H3.AE. H3.TAQ SYAMT (SEQ ID NO: AISGSGGNTFYAVSPIAAQMD For the following mAbs: 129) (Kabat); DSVKG (SEQ ID Y (SEQ IDL3.PY/H3.TAQ; GFTFGSY (SEQ ID NO: 132) (Kabat) NO: 135)L3.PY/L1.PS/H3.TAQ; NO: 130) (Chothia); SGSGGN (SEQ L3.PY/L1.AH/H3.TAQ;GFTFGSYAMT (SEQ ID NO: 133) L3.PY/L1.FF/H3.TAQ; ID NO: 131) (Chothia)L3.PY/L1.PH/H3.TAQ; (extended) and L3.PY/L3.KF/H3.TAQ. P5A2_VHVL andSYAMN (SEQ ID NO: AISDSGGSTYYA YWPMDI A02_Rd4_6nM_C03 150) (Kabat);DSVKG (SEQ ID NO: GFTFSSY (SEQ ID (SEQ ID NO: 153) 155) NO: 151)(Chothia); (Kabat) GFTFSSYAMN (SEQ SDSGGS (SEQ ID NO: 152) ID NO: 154)(extended) (Chothia) COMBO_Rd4_0.6nM_C17; SYPMS (SEQ ID NO: AIGGSGGSLPYAYWPMDI COMBO_Rd4_0.6nM_C14; 156) (Kabat); DSVKG (SEQ ID NO:COMBO_Rd4_0.6nM_C29; GFTFSSY (SEQ ID (SEQ ID NO: 158) 155) and NO: 151)(Chothia); (Kabat) COMBO_Rd4_0.6nM_C09 GFTFSSYPMS (SEQ GGSGGS (SEQ IDNO: 157) ID NO: 159) (extended) (Chothia) C01_Rd4_6nM_C04; SYPMS (SEQ IDNO: AIGGSGGSLPYA YWPMDS C01_Rd4_0.6nM_C03; 156) (Kabat); DSVKG (SEQ IDNO: C01_Rd4_0.6nM_C06; GFTFSSY (SEQ ID (SEQ ID NO: 158) 161)COMBO_Rd4_0.6nM_C02; NO: 151) (Chothia); (Kabat) COMBO_Rd4_6nM_C21;GFTFSSYPMS (SEQ GGSGGS (SEQ C01_Rd4_6nM_C26; ID NO: 157) ID NO: 159)COMBO_Rd4_0.6nM_C19; (extended) (Chothia) C01_Rd4_6nM_C24;C01_Rd4_6nM_C20; C01_Rd4_0.6nM_C09; COMBO_Rd4_0.6nM_C21;C01_Rd4_0.6nM_C04_C27; C01_Rd4_0.6nM_C16; C01_Rd4_6nM_C10;COMBO_Rd4_0.6nM_C20 P5C1_VHVL (PC1) and SYPMS (SEQ ID NO: AIGGSGGSTYYAYWPMDS COMBO_Rd4_0.6nM_C30 156) (Kabat); DSVKG (SEQ ID NO: GFTFSSY (SEQID (SEQ ID NO: 162) 161) NO: 151) (Chothia); (Kabat) GFTFSSYPMS (SEQGGSGGS (SEQ ID NO: 157) ID NO: 159) (extended) (Chothia)A02_Rd4_0.6nM_C06 SYAMN (SEQ ID NO: AISDSGGSAWY YWPMSL 150) (Kabat);ADSVKG (SEQ ID NO: GFTFSSY (SEQ ID (SEQ ID NO: 163) 164) NO: 151)(Chothia); (Kabat) GFTFSSYAMN (SEQ SDSGGS (SEQ ID NO: 152) ID NO: 154)(extended) (Chothia) A02_Rd4_0.6nM_C09 SYAMN (SEQ ID NO: AISDSGGSAWYYWPMSL 150) (Kabat); ADSVKG (SEQ ID NO: GFTFSSY (SEQ ID (SEQ ID NO: 163)164) NO: 151) (Chothia); (Kabat) GFTFSSYAMN (SEQ SDSGGS (SEQ ID NO: 152)ID NO: 154) (extended) (Chothia) A02_Rd4_0.6nM_C16; SYAMN (SEQ ID NO:AISDFGGSTYYA YWPMDI A02_Rd4_6nM_C16 150) (Kabat); DSVKG (SEQ ID NO:(P5A16) GFTFSSY (SEQ ID (SEQ ID NO: 165) 155) NO: 151) (Chothia);(Kabat) GFTFSSYAMN (SEQ SDFGGS (SEQ ID NO: 152) ID NO: 166) (extended)(Chothia) A02_Rd4_6nM_C01 SYAMN (SEQ ID NO: AITASGGSTYYA YWPMSL 150)(Kabat); DSVKG (SEQ ID NO: GFTFSSY (SEQ ID (SEQ ID NO: 167) 164) NO:151) (Chothia); (Kabat) GFTFSSYAMN (SEQ TASGGS (SEQ ID NO: 152) ID NO:168) (extended) (Chothia) A02_Rd4_6nM_C26 SYAMN (SEQ ID NO: AISDSGGSTYYAYWPMSL 150) (Kabat); DSVKG (SEQ ID NO: GFTFSSY (SEQ ID (SEQ ID NO: 153)164) NO: 151) (Chothia); (Kabat) GFTFSSYAMN (SEQ SDSGGS (SEQ ID NO: 152)ID NO: 154) (extended) (Chothia) A02_Rd4_6nM_C25 SYAMN (SEQ ID NO:AISDSGGSRWY YWPMTP 150) (Kabat); ADSVKG (SEQ ID NO: GFTFSSY (SEQ ID (SEQID NO: 169) 170) NO: 151) (Chothia); (Kabat) GFTFSSYAMN (SEQ SDSGGS (SEQID NO: 152) ID NO: 154) (extended) (Chothia) A02_Rd4_6nM_C22 SYAMN (SEQID NO: AVLDSGGSTYY YWPMTP 150) (Kabat); ADSVKG (SEQ ID NO: GFTFSSY (SEQID (SEQ ID NO: 171) 170) NO: 151) (Chothia); (Kabat) GFTFSSYAMN (SEQLDSGGS (SEQ ID NO: 152) ID NO: 172) (extended) (Chothia) A02_Rd4_6nM_C19SYAMN (SEQ ID NO: AISDSGGSRWY YWPMSD 150) (Kabat); ADSVKG (SEQ ID NO:GFTFSSY (SEQ ID (SEQ ID NO: 169) 173) NO: 151) (Chothia); (Kabat)GFTFSSYAMN (SEQ SDSGGS (SEQ ID NO: 152) ID NO: 154) (extended) (Chothia)A02_Rd4_0.6nM_C03 SYAMN (SEQ ID NO: AISDSGGSKWY YWPMSL 150) (Kabat);ADSVKG (SEQ (SEQ ID NO: GFTFSSY (SEQ ID ID NO: 174) 164) NO: 151)(Chothia); (Kabat) GFTFSSYAMN (SEQ SDSGGS (SEQ ID NO: 152) ID NO: 154)(extended) (Chothia) A02_Rd4_6nM_C07 SYAMN (SEQ ID NO: AIGGSGGSLPYAYWPMDS 150) (Kabat); DSVKG(SEQ ID (SEQ ID NO: GFTFSSY (SEQ ID NO: 158)(Kabat) 161) NO: 151) (Chothia); GGSGGS (SEQ GFTFSSYAMN (SEQ ID NO: 159)ID NO: 152) (Chothia) (extended) A02_Rd4_6nM_C23 SYAMN (SEQ ID NO:AISDSGGSGWY YWPMSL 150) (Kabat); ADSVKG (SEQ (SEQ ID NO: GFTFSSY (SEQ IDID NO: 175) 164) NO: 151) (Chothia); (Kabat) GFTFSSYAMN (SEQ SDSGGS (SEQID NO: 152) ID NO: 154) (extended) (Chothia) A02_Rd4_0.6nM_C18 SYAMN(SEQ ID NO: AVLDSGGSTYY YWPMSL 150) (Kabat); ADSVKG (SEQ ID NO: GFTFSSY(SEQ ID (SEQ ID NO: 171) 164) NO: 151) (Chothia); (Kabat) GFTFSSYAMN(SEQ LDSGGS (SEQ ID NO: 152) ID NO: 172) (extended) (Chothia)A02_Rd4_6nM_C10 SYAMN (SEQ ID NO: AISDSGGSCWY YWPMTP 150) (Kabat);ADSVKG (SEQ (SEQ ID NO: GFTFSSY (SEQ ID ID NO: 176) 170) NO: 151)(Chothia); (Kabat) GFTFSSYAMN (SEQ SDSGGS (SEQ ID NO: 152) ID NO: 154)(extended) (Chothia) A02_Rd4_6nM_C05 SYAMN (SEQ ID NO: AIFASGGSTYYAYWPMTP 150) (Kabat); DSVKG (SEQ ID NO: GFTFSSY (SEQ ID (SEQ ID NO: 177)170) NO: 151) (Chothia); (Kabat) GFTFSSYAMN (SEQ FASGGS (SEQ ID NO: 152)ID NO: 178) (extended) (Chothia) A02_Rd4_0.6nM_C10 SYAMN (SEQ ID NO:AISGWGGSLPY YWPMDS 150) (Kabat); ADSVKG (SEQ ID NO: GFTFSSY (SEQ ID (SEQID NO: 304) 161) NO: 151) (Chothia); (Kabat) GFTFSSYAMN (SEQ SGWGGS (SEQID NO: 152) ID NO: 179) (extended) (Chothia) A02_Rd4_6nM_C04 SYAMN (SEQID NO: AIMSSGGPLYYA YWPMAL 150) (Kabat); DSVKG (SEQ ID NO: GFTFSSY (SEQID (SEQ ID NO: 180) 182) NO: 151) (Chothia); (Kabat) GFTFSSYAMN (SEQMSSGGP (SEQ ID NO: 152) ID NO: 181) (extended) (Chothia)A02_Rd4_0.6nM_C26 SYAMN (SEQ ID NO: AILMSGGSTYYA YWPMSL 150) (Kabat);DSVKG (SEQ ID NO: GFTFSSY (SEQ ID (SEQ ID NO: 183) 164) NO: 151)(Chothia); (Kabat) GFTFSSYAMN (SEQ LMSGGS (SEQ ID NO: 152) ID NO: 184)(extended) (Chothia) A02_Rd4_0.6nM_C13 SYAMN (SEQ ID NO: AISDSGGYRYYAYWPMSL 150) (Kabat); DSVKG (SEQ ID NO: GFTFSSY (SEQ ID (SEQ ID NO: 185)164) NO: 151) (Chothia); (Kabat) GFTFSSYAMN (SEQ SDSGGY (SEQ ID NO: 152)ID NO: 186) (extended) (Chothia) A02_Rd4_0.6nM_C01 SYAMN (SEQ ID NO:AILSSGGSTYYA YWPMDI (P5AC1) 150) (Kabat); DSVKG (SEQ ID NO: GFTFSSY (SEQID (SEQ ID NO: 187) 155) NO: 151) (Chothia); (Kabat) GFTFSSYAMN (SEQLSSGGS (SEQ ID NO: 152) ID NO: 188) (extended) (Chothia) A02_Rd4_6nM_C08SYAMN (SEQ ID NO: AILDSGGSTYYA YWPMSP 150) (Kabat); DSVKG (SEQ ID (SEQID NO: GFTFSSY (SEQ ID NO: 160) (Kabat) 189) NO: 151) (Chothia); LDSGGS(SEQ GFTFSSYAMN (SEQ ID NO: 172) ID NO: 152) (Chothia) (extended)C01_Rd4_6nM_C12 SYPMS (SEQ ID NO: AIGGSGGWSYY YWPMDS (PC1C12) 156)(Kabat); ADSVKG (SEQ ID NO: GFTFSSY (SEQ ID (SEQ ID NO: 190) 161) NO:151) (Chothia); (Kabat) GFTFSSYPMS (SEQ GGSGGW (SEQ ID NO: 157) ID NO:191) (extended) (Chothia) C01_Rd4_6nM_C09 SYPMS (SEQ ID NO: ATVGSGGSIGYAYWPMDS 156) (Kabat); DSVKG (SEQ ID NO: GFTFSSY (SEQ ID (SEQ ID NO: 161)NO: 151) (Chothia); 192) (Kabat) GFTFSSYPMS (SEQ VGSGGS (SEQ ID NO: 157)ID NO: 193) (extended) (Chothia) COMBO_Rd4_0.6nM_C22 SYAMN (SEQ ID NO:AISDSGGSRWY YWPMDI (COM22) 150) (Kabat); ADSVKG (SEQ ID NO: GFTFSSY (SEQID (SEQ ID NO: 169) 155) NO: 151) (Chothia); (Kabat) GFTFSSYAMN (SEQSDSGGS (SEQ ID NO: 152) ID NO: 154) (extended) (Chothia)COMBO_Rd4_0.6nM_C10 SYPMS (SEQ ID NO: AIGGSGGSIHYA YWPMDS 156) (Kabat);DSVKG (SEQ ID (SEQ ID NO: GFTFSSY (SEQ ID NO: 194) (Kabat) 161) NO: 151)(Chothia); GGSGGS (SEQ GFTFSSYPMS (SEQ ID NO: 159) ID NO: 157) (Chothia)(extended) COMBO_Rd4_0.6nM_C04 SYPMS (SEQ ID NO: AHIGSGGSTYYA YWPMDS156) (Kabat); DSVKG (SEQ ID (SEQ ID NO: GFTFSSY (SEQ ID NO: 195) (Kabat)161) NO: 151) (Chothia); IGSGGS (SEQ ID GFTFSSYPMS (SEQ NO: 196) ID NO:157) (Chothia) (extended) COMBO_Rd4_0.6nM_C25 SYPMS (SEQ ID NO:AIGGSGGSTYYA YWPMDP 156) (Kabat); DSVKG (SEQ ID NO: GFTFSSY (SEQ ID (SEQID NO: 162) 197) NO: 151) (Chothia); (Kabat) GFTFSSYPMS (SEQ GGSGGS (SEQID NO: 157) ID NO: 159) (extended) (Chothia) COMBO_Rd4_6nM_C21 SYPMS(SEQ ID NO: AIGGSGGSLPYA YWPMDS 156) (Kabat); DSVKG (SEQ ID NO: GFTFSSY(SEQ ID (SEQ ID NO: 158) 161) NO: 151) (Chothia); (Kabat) GFTFSSYPMS(SEQ GGSGGS (SEQ ID NO: 157) ID NO: 159) (extended) (Chothia)COMBO_Rd4_6nM_C11 SYPMS (SEQ ID NO: AIGGSGGSLGYA YWPMDS 156) (Kabat);DSVKG (SEQ ID NO: GFTFSSY (SEQ ID (SEQ ID NO: 161) NO: 151) (Chothia);198)(Kabat) GFTFSSYPMS (SEQ GGSGGS (SEQ ID NO: 157) ID NO: 159)(extended) (Chothia) COMBO_Rd4_6nM_C09 SYPMS (SEQ ID NO: AIFASGGSTYYAYWPMDS 156) (Kabat); DSVKG (SEQ ID NO: GFTFSSY (SEQ ID (SEQ ID NO: 177)161) NO: 151) (Chothia); (Kabat) GFTFSSYPMS (SEQ FASGGS (SEQ ID NO: 157)ID NO: 178) (extended) (Chothia) COMBO_Rd4_6nM_C08 SYPMS (SEQ ID NO:AIGGSGTWTYY YWPMDS 156) (Kabat); ADSVKG (SEQ ID NO: GFTFSSY (SEQ ID (SEQID NO: 199) 161) NO: 151) (Chothia); (Kabat) GFTFSSYPMS (SEQ GGSGTW (SEQID NO: 157) ID NO: 200) (extended) (Chothia) COMBO_Rd4_0.6nM_C23 SYPMS(SEQ ID NO: ALFGSGGSTYY YWPMDS 156) (Kabat); ADSVKG (SEQ ID NO: GFTFSSY(SEQ ID (SEQ ID NO: 201) 161) NO: 151) (Chothia); (Kabat) GFTFSSYPMS(SEQ FGSGGS ID NO: 157) (SEQ ID NO: 202) (extended) (Chothia)COMBO_Rd4_0.6nM_C12 SYPMS (SEQ ID NO: AALGSGGSTYY YWPMDS 156) (Kabat);ADSVKG (SEQ (SEQ ID NO: GFTFSSY (SEQ ID ID NO: 203) 161) NO: 151)(Chothia); (Kabat) GFTFSSYPMS (SEQ LGSGGS (SEQ ID NO: 157) ID NO: 204)(extended) (Chothia) COMBO_Rd4_6nM_C07 SYPMS (SEQ ID NO: AIGGSGGSLPYAYWPMAD 156) (Kabat); DSVKG (SEQ ID NO: GFTFSSY (SEQ ID (SEQ ID NO: 158)205) NO: 151) (Chothia); (Kabat) GFTFSSYPMS (SEQ GGSGGS (SEQ ID NO: 157)ID NO: 159) (extended) (Chothia) COMBO_Rd4_6nM_C02 SYAMN (SEQ ID NO:AISDSGGFVYYA YWPMDS 150) (Kabat); DSVKG (SEQ ID NO: GFTFSSY (SEQ ID (SEQID NO: 206) 161) NO: 151) (Chothia); (Kabat) GFTFSSYAMN (SEQ SDSGGF (SEQID NO: 152) ID NO: 207) (extended) (Chothia) COMBO_Rd4_6nM_C05 SYAMN(SEQ ID NO: AIGGSGGSTYYA YWPMSL 150) (Kabat); DSVKG (SEQ ID NO: GFTFSSY(SEQ ID (SEQ ID NO: 162) 164) NO: 151) (Chothia); (Kabat) GFTFSSYAMN(SEQ GGSGGS (SEQ ID NO: 152) ID NO: 159) (extended) (Chothia)COMBO_Rd4_6nM_C22 SYAMN (SEQ ID NO: ACLDSGGSTYY YWPMDS 150) (Kabat);ADSVKG (SEQ (SEQ ID NO: GFTFSSY (SEQ ID ID NO: 208) 161) NO: 151)(Chothia); (Kabat) GFTFSSYAMN (SEQ LDSGGS (SEQ ID NO: 152) ID NO: 172)(extended) (Chothia) COMBO_Rd4_6nM_C11 SYPMS (SEQ ID NO: AALGSGGSTYYYWPMSL 156) (Kabat); ADSVKG (SEQ (SEQ ID NO: GFTFSSY (SEQ ID ID NO: 203)164) NO: 151) (Chothia); (Chothia) GFTFSSYPMS (SEQ LGSGGS (SEQ ID NO:157) ID NO: 204) (extended) (Chothia) Heavy chain consensus SYX₁MX₂,wherein X₁ AX₁X₂X₃X₄GX₅X₆ VSPIX₁X₂X₃ is A or P; and X₂ is T,X₇X₈YADX₉X₁₀KG, MDY, wherein N, or S (Kabat) (SEQ wherein X₁ is I, X₁ isA or Y; ID NO: 301) V, T, H, L, A, or X₂ is A or S; GFTFX₁SY, wherein C;X₂ is S, D, G, and X₃ is G, X₁ is G or S (Chothia) T, I, L, F, M, or V;Q, L, P, or E (SEQ ID NO: 302) X₃ is G, Y, L, H, (SEQ ID NO:GFTFX₁SYX₂MX₃, D, A, S, or M; X₄ 307) wherein X₁ is G or S, is S, Q, T,A, F, or YWPMX₁X₂, X₂ is A or P; and X₃ is W; X₅ is G or T; X₆ whereinX₁ is T, N, or S (SEQ ID is N, S, P, Y, W, D, S, T, or A; NO: 303)(extended) or F; X₇ is S, T, I, and X₂ is I, S, L, T, A, R, V, K, L, P,or D G, or C; X₈ is F, (SEQ ID NO: Y, P, W, H, or G; 308) X₉ is V, R, orL; and X₁₀ is G or T (Kabat) (SEQ ID NO: 305) X₁X₂X₃X₄X₅X₆, wherein X₁is S, V, I, D, G, T, L, F, or M; X₂ is G, Y, L, H, D, A, S, or M; X₃ isS, G, F, or W; X₄ is G or S; X₅ is G or T; and X₆ is N, S, P, Y, or W(Chothia) (SEQ ID NO: 306) P4G4 SYAMS (SEQ ID NO: SASGGS (SEQ LSWSGAFD381) (Kabat); ID NO: 383) N (SEQ ID GFTFSSY (SEQ ID (Chothia) NO: 385)NO: 151) (Chothia); AISASGGSTYYA GFTFSSYAMS (SEQ DSVKG (SEQ ID ID NO:382) NO: 384) (Kabat) (extended) P1A11 SYAMS (SEQ ID NO: SGSGGS (SEQVGTSGAFGI 386) (Kabat); ID NO: 389) (SEQ ID NO: GFTFRSY (SEQ ID(Chothia) 391) NO: 387) AISGSGGSTFYA GFTFRSYAMS (SEQ DSVKG (SEQ ID IDNO: 388) NO: 390) (Kabat) Light Chain mAb CDRL1 CDRL2 CDRL3 P6E01RASQSVSSSYLA GASSRAT (SEQ QHYGSPPSF For the following mAbs: (SEQ ID NO:209) ID NO: 210) T (SEQ ID P6E01/P6E01; and NO: 211) P6E01/H3.AQ.L1.LGF/L3.KW RASQSLGSFYLA GASSRAT (SEQ KHYGWPPS For the following mAbs:(SEQ ID NO: 212) ID NO: 210) FT (SEQ ID L1.LGF/L3.KW/P6E01; NO: 213)L1.LGF/L3.KW/H3.AL; L1.LGF/L3.KW/H3.AP; and L1.LGF/L3.KW/H3.AQL1.LGF/L3.NY RASQSLGSFYLA GASSRAT (SEQ QHYNYPPSF For the following mAbs:(SEQ ID NO: 212) ID NO: 210) T (SEQ ID L1.LGF/L3.NY/P6E01; NO: 214)L1.LGF/L3.NY/H3.AL; L1.LGF/L3.NY/H3.AP; and L1.LGF/L3.NY/H3AQL1.GDF/L3.NY RASQSVGDFYLA GASSRAT (SEQ QHYNYPPSF For the following mAbs:(SEQ ID NO: 215) ID NO: 210) T (SEQ ID L1.GDF/L3.NY/P6E01; NO: 214)L1.GDF/L3.NY/H3.AL; L1.GDF/L3.NY/H3.AP; and L1.GDF/L3.NY/H3.AQL1.LGF/L3.PY RASQSLGSFYLA GASSRAT (SEQ QHYPYPPSFT For the followingmAbs: (SEQ ID NO: 212) ID NO: 210) (SEQ ID NO: L1.LGF/L3.PY/H3.AP; 216)and L1.LGF/L3.PY/H3.AQ L1.GDF/L3.KW RASQSVGDFYLA GASSRAT (SEQ KHYGWPPSFor the following mAbs: (SEQ ID NO: 215) ID NO: 210) FT (SEQ IDL1.GDF/L3.KW/H3.AL; NO: 213) L1.GDF/L3.KW/H3.AP; and L1.GDF/ L3.KW/H3.AQL1.GDF/L3.PY/H3.AQ RASQSVGDFYLA GASSRAT (SEQ QHYPYPPSF (SEQ ID NO: 215)ID NO: 210) T (SEQ ID NO: 216) L3.KW/P6E01 RASQSVSSSYLA GASSRAT (SEQKHYGWPPS (SEQ ID NO: 209) ID NO: 210) FT (SEQ ID NO: 213) L3.PYRASQSVSSSYLA GASSRAT (SEQ QHYPYPPSFT For the following mAbs: (SEQ ID NO:209) ID NO: 210) (SEQ ID NO: L3.PY/P6E01; 216) L3.PY/H2.QR; L3.PY/H2.DY;L3.PY/H2.YQ; L3.PY/H2.LT; L3.PY/H2.HA; L3.PY/H2.QL; L3.PY/H3.YA;L3.PY/H3.AE; L3.PY/H3.AQ; L3.PY/H3.TAQ L3.NY/P6E01 RASQSVSSSYLA GASSRAT(SEQ QHYNYPPSFT (SEQ ID NO: 209) ID NO: 210) (SEQ ID NO: 214)L3.PY/L1.PS RASQSVSSSYPS GASSRAT (SEQ QHYPYPPSFT For the following mAbs:(SEQ ID NO: 217) ID NO: 210) (SEQ ID NO: L3.PY/L1.PS/P6E01; 216) P6DY;L3.PY/L1.PS/H2.Q R; L3.PY/L1.PS/H2.DY; L3.PY/L1.PS/H2.YQ;L3.PY/L1.PS/H2.LT; L3.PY/L1.PS/H2.HA; L3.PY/L1.PS/H2.QL;L3.PY/L1.PS/H3.YA; L3.PY/L1.PS/H3.AE; L3.PY/L1.PS/H3.AQ;L3.PY/L1.PS/H3.TAQ; L3.PY/L1.AH RASQSVSAHYLA GASSRAT (SEQ QHYPYPPSFT Forthe following mAbs: (SEQ ID NO: 218) ID NO: 210) (SEQ ID NO:L3.PY/L1.AH/P6E01; 216) L3.PY/L1.AH/H2.QR; L3.PY/L1.AH/H2.DY;L3.PY/L1.AH/H2.YQ; L3.PY/L1.AH/H2.LT; L3.PY/L1.AH/H2.HA;L3.PY/L1.AH/H2.QL; L3.PY/L1.AH/H3.YA; L3.PY/L1.AH/H3.AE;L3.PY/L1.AH/H3.AQ; L3.PY/L1.AH/H3.TAQ L3.PY/L1.FF RASQSVSSFFLA GASSRAT(SEQ QHYPYPPSFT For the following mAbs: (SEQ ID NO: 219) ID NO: 210)(SEQ ID NO: L3.PY/L1.FF/P6E01; 216) L3.PY/L1.FF/H2.QR;L3.PY/L1.FF/H2.DY; L3.PY/L1.FF/H2.YQ; L3.PY/L1.FF/H2.LT;L3.PY/L1.FF/H2.HA; L3.PY/L1.FF/H2.QL; L3.PY/L1.FF/H3.YA;L3.PY/L1.FF/H3.AE; L3.PY/L1.FF/H3.AQ; and L3.PY/L1.FF/H3.TAQ L3.PY/L1.PHRASQSVSPHYLA GASSRAT (SEQ QHYPYPPSFT For the following mAbs: (SEQ ID NO:219) ID NO: 210) (SEQ ID NO: L3.PY/L1.PH/P6E01; 216) L3.PY/L1.PH/H2.QR;L3.PY/L1.PH/H2.HA; L3.PY/L1.PH/H3.AE; L3.PY/L1.PH/H3.AQ; andL3.PY/L1.PH/H3.TAQ L3.PY/L3.KY RASQSVSSSYLA GASSRAT (SEQ KYYPYPPSFT Forthe following mAbs: (SEQ ID NO: 209) ID NO: 210) (SEQ ID NO:L3.PY/L3.KY/P6E01; 220) L3.PY/L3.KY/H2.QR; L3.PY/L3.KY/H2.DY;L3.PY/L3.KY/H2.YQ; L3.PY/L3.KY/H2.LT; L3.PY/L3.KY/H2.HA;L3.PY/L3.KY/H2.QL; L3.PY/L3.KY/H3.YA; and L3.PY/L3.KY/H3.TAQ L3.PY/L3.KFRASQSVSSSYLA GASSRAT (SEQ KFYPYPPSF For the following mAbs: (SEQ ID NO:209) ID NO: 210) T (SEQ ID L3.PY/L3.KF/H2.DY; NO: 220)L3.PY/L3.KF/H2.YQ; L3.PY/L3.KF/H2.LT; L3.PY/L3.KF/H2.QL;L3.PY/L3.KF/H3.YA; L3.PY/L3.KF/H3.AE; L3.PY/L3.KF/H3.AQ; andL3.PY/L3.KF/H3.TAQ P5A2_VHVL (P5A) RASQSVSSSYLA DASIRAT QQYGSWPL (SEQ IDNO: 209) (SEQ ID NO: 221) T (SEQ ID NO: 222) A02_Rd4_0.6nM_C06RASQSVSVIYLA DASIRAT QQYQRWPLT (SEQ ID NO: 223) (SEQ ID NO: 221) (SEQ IDNO: 224) A02_Rd4_0.6nM_C09; RASQSVSSSYLA DASIRAT QQYQSWPLTCOMBO_Rd_0.6nM_C29; (SEQ ID NO: 209) (SEQ ID NO: 221) (SEQ ID NO: and225) COMBO_Rd4_0.6nM_C21 A02_Rd4_6nM_C16 RASQSVSDIYLA DASIRAT QQYQTWPL(P5AC16) (SEQ ID NO: 226) (SEQ ID NO: 221) T (SEQ ID NO: 227)A02_Rd4_6nM_C03 RASQSVSNIYLA DASIRAT QQYQGWPL (SEQ ID NO: 228) (SEQ IDNO: 221) T (SEQ ID NO: 229) A02_Rd4_6nM_C01 RASQSVSAYYLA DASIRATQQYERWPLT (SEQ ID NO: 230) (SEQ ID NO: 221) (SEQ ID NO: 231)A02_Rd4_6nM_C26 RASQSVSSIYLA DASIRAT QQYQVWPLT (SEQ ID NO: 232) (SEQ IDNO: 221) (SEQ ID NO: 233) A02_Rd4_6nM_C25 RASQSVSSSYLA DASIRAT QQYLDWPLT(SEQ ID NO: 209) (SEQ ID NO: 221) (SEQ ID NO: 234) A02_Rd4_6nM_C22RASQSVSSSYLA DASIRAT QQYQVWPLT (SEQ ID NO: 209) (SEQ ID NO: 221) (SEQ IDNO: 233) A02_Rd4_6nM_C19 RASQSVSVIYLA DASIRAT QQYLAWPLT (SEQ ID NO: 223)(SEQ ID NO: 221) (SEQ ID NO: 236) A02_Rd4_0.6nM_C03 RASQSVSSSYLA DASIRATQQYFTWPLT (SEQ ID NO: 209) (SEQ ID NO: 221) (SEQ ID NO: 237)A02_Rd4_6nM_C07 RASQSVSPYYLA DASIRAT QQYERWPLT (SEQ ID NO: 238) (SEQ IDNO: 221) (SEQ ID NO: 231) A02_Rd4_6nM_C23 RASQSVSVEYLA DASIRAT QQYARWPLT(SEQ ID NO: 239) (SEQ ID NO: 221) (SEQ ID NO: 240) A02_Rd4_0.6nM_C18RASQSVSEIYLA DASIRAT QQYFGWPLT (SEQ ID NO: 241) (SEQ ID NO: 221) (SEQ IDNO: 242) A02_Rd4_6nM_C10 RASQSVEMSYLA DASIRAT QQYAHWPLT (SEQ ID NO: 243)(SEQ ID NO: 221) (SEQ ID NO: 244) A02_Rd4_6nM_C05 RASQSVSSSYLA DASIRATQQYQRWPLT (SEQ ID NO: 209) (SEQ ID NO: 221) (SEQ ID NO: 224)A02_Rd4_0.6nM_C10 RASQSVSAQYLA DASIRAT QQYQRWPLT (SEQ ID NO: 245) (SEQID NO: 221) (SEQ ID NO: 224) A02_Rd4_6nM_C04 RASQSVSAIYLA DASIRATQQYQVWPLT (SEQ ID NO: 235) (SEQ ID NO: 221) (SEQ ID NO: 233)A02_Rd4_0.6nM_C26 GPSQSVSSSYLA DASIRAT QQYQSWPLT (SEQ ID NO: 246) (SEQID NO: 221) (SEQ ID NO: 225) A02_Rd4_0.6nM_C13 RASQSVSSSYWA DASIRATQQYESWPLT (SEQ ID NO: 247) (SEQ ID NO: 221) (SEQ ID NO: 248)A02_Rd4_0.6nM_C01 RGGQSVSSSYLA DASIRAT QQYQSWPLT (P5AC1) (SEQ ID NO:249) (SEQ ID NO: 221) (SEQ ID NO: 225) A02_Rd4_6nM_C08 RASQSVSFIYLADASIRAT QQYGSWPL (SEQ ID NO: 250) (SEQ ID NO: 221) T (SEQ ID NO: 222)P5C1_VHVL (PC1) RASQSVSSTYLA DASSRAP QQYSTSPLT (SEQ ID NO: 251) (SEQ IDNO: 252) (SEQ ID NO: 253) C01_Rd4_6nM_C24 RASQSVSPEYLA DASSRAP QQYSVWPLT(SEQ ID NO: 254) (SEQ ID NO: 252) (SEQ ID NO: 255) C01_Rd4_6nM_C26RASQSVSAIYLA DASSRAP QQYSAWPLT (SEQ ID NO: 235) (SEQ ID NO: 252) (SEQ IDNO: 256) C01_Rd4_6nM_C10 RASQSVSSVYLA DASSRAP QQYSTWPLT (SEQ ID NO: 257)(SEQ ID NO: 252) (SEQ ID NO: 258) C01_Rd4_0.6nM_C27 RASQSVSSTYLA DASSRAPQQYSRWPLT (SEQ ID NO: 251) (SEQ ID NO: 252) (SEQ ID NO: 259)C01_Rd4_6nM_C20 RASQSVSPIYLA DASSRAP QQYSAFPLT (SEQ ID NO: 260) (SEQ IDNO: 252) (SEQ ID NO: 261) C01_Rd4_6nM_C12 WLSQSVSSTYLA DASSRAP QQYSEWPLT(PC1C12) (SEQ ID NO: 262) (SEQ ID NO: 252) (SEQ ID NO: 263)C01_Rd4_0.6nM_C16 RASQSVSSTYLA DASSRAP QQYSSWPLT (SEQ ID NO: 251) (SEQID NO: 252) (SEQ ID NO: 264) C01_Rd4_0.6nM_C09 RASQSVSSIFLA DASSRAPQQYSAWPLT (SEQ ID NO: 265) (SEQ ID NO: 252) (SEQ ID NO: 256)C01_Rd4_6nM_C09 ACSQSVSSTYLA DASSRAP QQYSAWPLT (SEQ ID NO: 266) (SEQ IDNO: 252) (SEQ ID NO: 256) C01_Rd4_0.6nM_C03 RASCDVSSTYLA DASSRAPQQYMRSPLT (SEQ ID NO: 267) (SEQ ID NO: 252) (SEQ ID NO: 268)C01_Rd4_0.6nM_C06 RASEAVPSTYLA DASSRAP QQYSAFPLT (SEQ ID NO: 269) (SEQID NO: 252) (SEQ ID NO: 261) C01_Rd4_0.6nM_C04 CSSQSVSSTYLA DASSRAPQQYSAFPLT (SEQ ID NO: 270) (SEQ ID NO: 252) (SEQ ID NO: 261)COMBO_Rd4_0.6nM_C22 RASVRVSSTYLA DASIRAT QQYMKWPLT (COM22) (SEQ ID NO:271) (SEQ ID NO: 221) (SEQ ID NO: 272) COMBO_Rd4_6nM_C21 RASQSVSAAYLADASIRAT QQYMCWPLT (SEQ ID NO: 273) (SEQ ID NO: 221) (SEQ ID NO: 274)COMBO_Rd4_6nM_C10 RASQSVSSSYWG DASIRAT QQYQCWPLT (SEQ ID NO: 275) (SEQID NO: 221) (SEQ ID NO: 276) COMBO_Rd4_0.6nM_C04 RASQSVSSTYLA DASIRATQQYQSWPLT (SEQ ID NO: 251) (SEQ ID NO: 221) (SEQ ID NO: 225)COMBO_Rd4_6nM_C25 RASQSVSSPYLA DASIRAT QQYQSWPLT (SEQ ID NO: 277) (SEQID NO: 221) (SEQ ID NO: 225) COMBO_Rd4_6nM_C11 RASQSVSPIYLA DASIRATQQYKAWPLT (SEQ ID NO: 260) (SEQ ID NO: 221) (SEQ ID NO: 278)COMBO_Rd4_0.6nM_C20 RASQSVSYLYLA DASIRAT QQYMEWPLT (SEQ ID NO: 279) (SEQID NO: 221) (SEQ ID NO: 280) COMBO_Rd4_6nM_C09 RASQSVSAQYLA DASIRATQQYQAWPLT (SEQ ID NO: 245) (SEQ ID NO: 221) (SEQ ID NO: 281)COMBO_Rd4_6nM_C08 RASQSVSSSYLA DASIRAT QQYQKWPLT (SEQ ID NO: 209) (SEQID NO: 221) (SEQ ID NO: 282) COMBO_Rd4_0.6nM_C19 RASQSVSAVYLA DASIRATQQYRAWPLT (SEQ ID NO: 283) (SEQ ID NO: 221) (SEQ ID NO: 284)COMBO_Rd4_0.6nM_C02 RASIAVSSTYLA DASIRAT QQYMVWPLT (SEQ ID NO: 285) (SEQID NO: 221) (SEQ ID NO: 286) COMBO_Rd4_0.6nM_C23 RPRQSVSSSYLA DASIRATQQYQDWPLT (SEQ ID NO: 287) (SEQ ID NO: 221) (SEQ ID NO: 288)COMBO_Rd4_0.6nM_C09 RASQSVSSTYLA DASIRAT QQYQEWPLT (SEQ ID NO: 251) (SEQID NO: 221) (SEQ ID NO: 289) COMBO_Rd4_6nM_C12 RASQSVSASYLA DASIRATQQYMSWPLT (SEQ ID NO: 290) (SEQ ID NO: 221) (SEQ ID NO: 291)COMBO_Rd4_0.6nM_C30 RASQSVSYMYLA DASIRAT QQYKSWPLT (SEQ ID NO: 292) (SEQID NO: 221) (SEQ ID NO: 293) COMBO_Rd4_0.6nM_C14 RASQSVSAIYLA DASIRATQQYYGWPLT (SEQ ID NO: 235) (SEQ ID NO: 221) (SEQ ID NO: 294)COMBO_Rd4_6nM_C07 RASQPISSSYLA DASIRAT QQYQGWPLT (SEQ ID NO: 295) (SEQID NO: 221) (SEQ ID NO: 229) COMBO_Rd4_6nM_C02 RASQSVSSSYLA DASIRATQQYEFWPLT (SEQ ID NO: 209) (SEQ ID NO: 221) (SEQ ID NO: 296)COMBO_Rd4_0.6nM_C05 RASQSVSSTYLA DASIRAT QQYMSWPLT (SEQ ID NO: 251) (SEQID NO: 221) (SEQ ID NO: 291) COMBO_Rd4_0.6nM_C17 RASQGISSTYLA DASIRATQQYAYWPLT (SEQ ID NO: 297) (SEQ ID NO: 221) (SEQ ID NO: 298)COMBO_Rd4_6nM_C22 RASQSVSSSYLA DASIRAT QQYQGWPLT (SEQ ID NO: 209) (SEQID NO: 221) (SEQ ID NO: 229) COMBO_Rd4_0.6nM_C11 RASQSVSVRYLA DASIRATQQYGSWPIT (SEQ ID NO: 299) (SEQ ID NO: 221) (SEQ ID NO: 300) Light chainconsensus X₁X₂X₃X₄X₅X₆X₇X₈X₉ X₁ASX₂RAX₃, X₁X₂YX₃X₄PP X₁₀X₁₁X₁₂, whereinX₁ wherein X₁ is G or SFT, wherein is R, G, W, A, or C; X₂ D; X₂ is S orI; X₁ is Q or K; is A, P, G, L, C, or S; and X₃ is T or P X₂ is H or Y;X₃ is S, G, or R; X₄ is (SEQ ID NO: 310) X₃ is G, N, or Q, C, E, V, orI; X₅ is P; and X₄ is S, P, G, A, R, or D; X₆ S, W, or Y is V, G, I, orL; X₇ is S, (SEQ ID NO: E, D, P, or G; X₈ is S, 311) P, F, A, M, E, V,N, D, QQYX₁X₂X₃P or Y; X₉ is I, T, V, E, F X₄T, wherein S, A, M, Q, Y,H, or R; X₁ is G, Q, E, X₁₀ is Y or F; X₁₁ is L, L, F, A, S, M, W, or P;and X₁₂ is A, K, R, or Y; X₂ S, or G (SEQ ID NO: is S, R, T, G, 309) V,F, Y, D, A, H, V, E, K, or C; X₃ is W, F, or S; and X₄ is L or I (SEQ IDNO: 312) P4G4 RASQSVSSSYLA GASSRAY (SEQ QHYGSPPLF (SEQ ID NO: 209) IDNO: 392) T (SEQ ID NO: 393) P1A11 RASQNVSSSYLA GASYRAT (SEQ QHYGSPPSF(SEQ ID NO: 379) ID NO: 395) T (SEQ ID NO: 211) P6AP RASQLGSFYLA GASSRAT(SEQ QHYNYPPSF (SEQ ID NO: 377) ID NO: 210) T (SEQ ID NO: 214)

The invention encompasses modifications to the CARs and polypeptides ofthe invention variants shown in Table 1, including functionallyequivalent CARs having modifications which do not significantly affecttheir properties and variants which have enhanced or decreased activityand/or affinity. For example, the amino acid sequence may be mutated toobtain an antibody with the desired binding affinity to BCMA.Modification of polypeptides is routine practice in the art and need notbe described in detail herein. Examples of modified polypeptides includepolypeptides with conservative substitutions of amino acid residues, oneor more deletions or additions of amino acids which do not significantlydeleteriously change the functional activity, or which mature (enhance)the affinity of the polypeptide for its ligand, or use of chemicalanalogs.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto an epitope tag. Other insertional variants of the antibody moleculeinclude the fusion to the N- or C-terminus of the antibody of an enzymeor a polypeptide which increases the half-life of the antibody in theblood circulation.

Substitution variants have at least one amino acid residue in theantibody molecule removed and a different residue inserted in its place.The sites of greatest interest for substitutional mutagenesis includethe hypervariable regions, but FR alterations are also contemplated.Conservative substitutions are shown in Table 2.1 under the heading of“conservative substitutions.” If such substitutions result in a changein biological activity, then more substantial changes, denominated“exemplary substitutions” in Table 2.1, or as further described below inreference to amino acid classes, may be introduced and the productsscreened.

TABLE 2.1 Amino Acid Substitutions Original Residue (naturally occurringamino Conservative acid) Substitutions Exemplary Substitutions Ala (A)Val Val; Leu; Ile Arg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Asp,Lys; Arg Asp (D) Glu Glu; Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn; GluGlu (E) Asp Asp; Gln Gly (G) Ala Ala His (H) Arg Asn; Gln; Lys; Arg Ile(I) Leu Leu; Val; Met; Ala; Phe; Norleucine Leu (L) Ile Norleucine; Ile;Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; IlePhe (F) Tyr Leu; Val; Ile; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr(T) Ser Ser Trp (W) Tyr Tyr; Phe Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V)Leu Ile; Leu; Met; Phe; Ala; Norleucine

In some embodiments, the invention provides a CAR comprising anextracellular ligand-binding domain that binds to BCMA and competes forbinding to BCMA with a CAR described herein, including P6E01/P6E01,P6E01/H3.AQ, L1.LGF/L3.KW/P6E01; L1.LGF/L3.NY/P6E01, L1.GDF/L3.NY/P6E01,L1.LGF/L3.KW/H3.AL, L1.LGF/L3.KW/H3.AP, L1.LGF/L3.KW/H3.AQ,L1.LGF/L3.PY/H3.AP, L1.LGF/L3.PY/H3.AQ, L1.LGF/L3.NY/H3.AL,L1.LGF/L3.NY/H3.AP, L1.LGF/L3.NY/H3.AQ, L1.GDF/L3.KW/H3.AL,L1.GDF/L3.KW/H3.AP, L1.GDF/L3.KW/H3.AQ, L1.GDF/L3.PY/H3.AQ,L1.GDF/L3.NY/H3.AL, L1.GDF/L3.NY/H3.AP, L1.GDF/L3.NY/H3.AQ, L3.KW/P6E01,L3.PY/P6E01, L3.NY/P6E01, L3.PY/L1.PS/P6E01, L3.PY/L1.AH/P6E01,L3.PY/L1.FF/P6E01, L3.PY/L1.PH/P6E01, L3.PY/L3.KY/P6E01,L3.PY/L3.KF/P6E01, L3.PY/H2.QR, L3.PY/H2.DY, L3.PY/H2.YQ, L3.PY/H2.LT,L3.PY/H2.HA, L3.PY/H2.QL, L3.PY/H3.YA, L3.PY/H3.AE, L3.PY/H3.AQ,L3.PY/H3.TAQ, L3.PY/P6E01, L3.PY/L1.PS/H2.QR, L3.PY/L1.PS/H2.DY,L3.PY/L1.PS/H2.YQ, L3.PY/L1.PS/H2.LT, L3.PY/L1.PS/H2.HA,L3.PY/L1.PS/H2.QL, L3.PY/L1.PS/H3.YA, L3.PY/L1.PS/H3.AE,L3.PY/L1.PS/H3.AQ, L3.PY/L1.PS/H3.TAQ, L3.PY/L1.AH/H2.QR,L3.PY/L1.AH/H2.DY, L3.PY/L1.AH/H2.YQ, L3.PY/L1.AH/H2.LT,L3.PY/L1.AH/H2.HA, L3.PY/L1.AH/H2.QL, L3.PY/L1.AH/H3.YA,L3.PY/L1.AH/H3.AE, L3.PY/L1.AH/H3.AQ, L3.PY/L1.AH/H3.TAQ,L3.PY/L1.FF/H2.QR, L3.PY/L1.FF/H2.DY, L3.PY/L1.FF/H2.YQ,L3.PY/L1.FF/H2.LT, L3.PY/L1.FF/H2.HA, L3.PY/L1.FF/H2.QL,L3.PY/L1.FF/H3.YA, L3.PY/L1.FF/H3.AE, L3.PY/L1.FF/H3.AQ,L3.PY/L1.FF/H3.TAQ, L3.PY/L1.PH/H2.QR, L3.PY/L1.PH/H2.HA,L3.PY/L1.PH/H3.AE, L3.PY/L1.PH/H3.AQ, L3.PY/L1.PH/H3.TAQ,L3.PY/L3.KY/H2.QR, L3.PY/L3.KY/H2.DY, L3.PY/L3.KY/H2.YQL3.PY/L3.KY/H2.LT, L3.PY/L3.KY/H2.HA, L3.PY/L3.KY/H2.QL,L3.PY/L3.KY/H3.YA L3.PY/L3.KY/H3.TAQ, L3.PY/L3.KF/H2.DY,L3.PY/L3.KF/H2.YQ, L3.PY/L3.KF/H2.LT L3.PY/L3.KF/H2.QL,L3.PY/L3.KF/H3.YA, L3.PY/L3.KF/H3.AE, L3.PY/L3.KF/H3.AQL3.PY/L3.KF/H3.TAQ, P5A2_VHVL, A02_Rd4_0.6nM_C06, A02_Rd4_0.6nM_C09A02_Rd4_6nM_C16, A02_Rd4_6nM_C03, A02_Rd4_6nM_C01, A02_Rd4_6nM_C26A02_Rd4_6nM_C25, A02_Rd4_6nM_C22, A02_Rd4_6nM_C19, A02_Rd4_0.6nM_C03A02_Rd4_6nM_C07, A02_Rd4_6nM_C23, A02_Rd4_0.6nM_C18, A02_Rd4_6nM_C10A02_Rd4_6nM_C05, A02_Rd4_0.6nM_C10, A02_Rd4_6nM_C04, A02_Rd4_0.6nM_C26A02_Rd4_0.6nM_C13, A02_Rd4_0.6nM_C01, A02_Rd4_6nM_C08, P5C1_VHVL,C01_Rd4_6nM_C24, C01_Rd4_6nM_C26, C01_Rd4_6nM_C10, C01_Rd4_0.6nM_C27C01_Rd4_6nM_C20, C01_Rd4_6nM_C12, C01_Rd4_0.6nM_C16, C01_Rd4_0.6nM_C09C01_Rd4_6nM_C09, C01_Rd4_0.6nM_C03, C01_Rd4_0.6nM_C06, C01_Rd4_6nM_C04COMBO_Rd4_0.6nM_C22, COMBO_Rd4_6nM_C21, COMBO_Rd4_6nM_C10,COMBO_Rd4_0.6nM_C04, COMBO_Rd4_6nM_C25, COMBO_Rd4_0.6nM_C21,COMBO_Rd4_6nM_C11, COMBO_Rd4_0.6nM_C20, COMBO_Rd4_6nM_C09,COMBO_Rd4_6nM_C08, COMBO_Rd4_0.6nM_C19, COMBO_Rd4_0.6nM_C02,COMBO_Rd4_0.6nM_C23, COMBO_Rd4_0.6nM_C29, COMBO_Rd4_0.6nM_C09,COMBO_Rd4_6nM_C12, COMBO_Rd4_0.6nM_C30, COMBO_Rd4_0.6nM_C14,COMBO_Rd4_6nM_C07, COMBO_Rd4_6nM_C02, COMBO_Rd4_0.6nM_C05,COMBO_Rd4_0.6nM_C17, COMBO_Rd4_6nM_C22, COMBO_Rd4_0.6nM_C11, orCOMBO_Rd4_0.6nM_C29.

In some embodiments, the invention provides a CAR, which specificallybinds to BCMA, wherein the CAR comprises a VH region comprising asequence shown in SEQ ID NO: 33; and/or a VL region comprising asequence shown in SEQ ID NO: 34. In some embodiments, the inventionprovides a CAR, which specifically binds to BCMA, wherein the CARcomprises a VH region comprising a sequence shown in SEQ ID NO: 33, 72,39, 76, 83, 92, 25, or 8; and/or a VL region comprising a sequence shownin SEQ ID NO: 34, 73, 40, 77, 84, 93, 18, or 80. In some embodiments,the invention also provides CARs comprising CDR portions of antibodiesto BCMA antibodies based on CDR contact regions. CDR contact regions areregions of an antibody that imbue specificity to the antibody for anantigen. In general, CDR contact regions include the residue positionsin the CDRs and Vernier zones which are constrained in order to maintainproper loop structure for the antibody to bind a specific antigen. See,e.g., Makabe et al., J. Biol. Chem., 283:1156-1166, 2007. Determinationof CDR contact regions is well within the skill of the art. The bindingaffinity (K_(D)) of the BCMA specific CAR as described herein to BCMA(such as human BCMA (e.g., (SEQ ID NO: 354) can be about 0.002 to about6500 nM. In some embodiments, the binding affinity is about any of 6500nm, 6000 nm, 5986 nm, 5567 nm, 5500 nm, 4500 nm, 4000 nm, 3500 nm, 3000nm, 2500 nm, 2134 nm, 2000 nm, 1500 nm, 1000 nm, 750 nm, 500 nm, 400 nm,300 nm, 250 nm, 200 nM, 193 nM, 100 nM, 90 nM, 50 nM, 45 nM, 40 nM, 35nM, 30 nM, 25 nM, 20 nM, 19 nm, 18 nm, 17 nm, 16 nm, 15 nM, 10 nM, 8 nM,7.5 nM, 7 nM, 6.5 nM, 6 nM, 5.5 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5nM, 0.3 nM, 0.1 nM, 0.01 nM, or 0.002 nM. In some embodiments, thebinding affinity is less than about any of 6500 nm, 6000 nm, 5500 nm,5000 nm, 4000 nm, 3000 nm, 2000 nm, 1000 nm, 900 nm, 800 nm, 250 nM, 200nM, 100 nM, 50 nM, 30 nM, 20 nM, 10 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, or 0.5 nM.

The intracellular signaling domain of a CAR according to the inventionis responsible for intracellular signaling following the binding ofextracellular ligand-binding domain to the target resulting in theactivation of the immune cell and immune response. The intracellularsignaling domain has the ability to activate of at least one of thenormal effector functions of the immune cell in which the CAR isexpressed. For example, the effector function of a T cell can be acytolytic activity or helper activity including the secretion ofcytokines.

In some embodiments, an intracellular signaling domain for use in a CARcan be the cytoplasmic sequences of, for example without limitation, theT cell receptor and co-receptors that act in concert to initiate signaltransduction following antigen receptor engagement, as well as anyderivative or variant of these sequences and any synthetic sequence thathas the same functional capability. Intracellular signaling domainscomprise two distinct classes of cytoplasmic signaling sequences: thosethat initiate antigen-dependent primary activation, and those that actin an antigen-independent manner to provide a secondary orco-stimulatory signal. Primary cytoplasmic signaling sequences cancomprise signaling motifs which are known as immunoreceptortyrosine-based activation motifs of ITAMs. ITAMs are well definedsignaling motifs found in the intracytoplasmic tail of a variety ofreceptors that serve as binding sites for syk/zap70 class tyrosinekinases. Examples of ITAM used in the invention can include as nonlimiting examples those derived from TCRζ, FcRγ, FcRβ, FcRβ, CD3γ, CD3δ,CD3ε, CD5, CD22, CD79a, CD79b and CD66d. In some embodiments, theintracellular signaling domain of the CAR can comprise the CD3ζsignaling domain which has amino acid sequence with at least about 70%,preferably at least 80%, more preferably at least 90%, 95% 97%, or 99%sequence identity with an amino acid sequence shown in SEQ. ID NO: 324.In some embodiments the intracellular signaling domain of the CAR of theinvention comprises a domain of a co-stimulatory molecule.

In some embodiments, the intracellular signaling domain of a CAR of theinvention comprises a part of co-stimulatory molecule selected from thegroup consisting of fragment of 41BB (GenBank: AAA53133.) and CD28(NP_006130.1). In some embodiments, the intracellular signaling domainof the CAR of the invention comprises amino acid sequence whichcomprises at least 70%, preferably at least 80%, more preferably atleast 90%, 95%, 97%, or 99% sequence identity with an amino acidsequence shown in SEQ. ID NO: 323 and SEQ. ID NO: 327.

CARs are expressed on the surface membrane of the cell. Thus, the CARcan comprise a transmembrane domain. Suitable transmembrane domains fora CAR disclosed herein have the ability to (a) be expressed at thesurface of a cell, preferably an immune cell such as, for examplewithout limitation, lymphocyte cells or Natural killer (NK) cells, and(b) interact with the ligand-binding domain and intracellular signalingdomain for directing cellular response of immune cell against apredefined target cell. The transmembrane domain can be derived eitherfrom a natural or from a synthetic source. The transmembrane domain canbe derived from any membrane-bound or transmembrane protein. Asnon-limiting examples, the transmembrane polypeptide can be a subunit ofthe T cell receptor such as α, β, γ or δ, polypeptide constituting CD3complex, IL-2 receptor p55 (a chain), p75 (β chain) or γ chain, subunitchain of Fc receptors, in particular Fcγ receptor III or CD proteins.Alternatively, the transmembrane domain can be synthetic and cancomprise predominantly hydrophobic residues such as leucine and valine.In some embodiments said transmembrane domain is derived from the humanCD8α chain (e.g., NP_001139345.1). The transmembrane domain can furthercomprise a stalk domain between the extracellular ligand-binding domainand said transmembrane domain. A stalk domain may comprise up to 300amino acids, preferably 10 to 100 amino acids and most preferably 25 to50 amino acids. Stalk region may be derived from all or part ofnaturally occurring molecules, such as from all or part of theextracellular region of CD8, CD4, or CD28, or from all or part of anantibody constant region. Alternatively the stalk domain may be asynthetic sequence that corresponds to a naturally occurring stalksequence, or may be an entirely synthetic stalk sequence. In someembodiments said stalk domain is a part of human CD8α chain (e.g.,NP_001139345.1). In another particular embodiment, said transmembraneand hinge domains comprise a part of human CD8α chain, preferably whichcomprises at least 70%, preferably at least 80%, more preferably atleast 90%, 95% 97%, or 99% sequence identity with amino acid sequenceselected from the group consisting of SEQ ID NO: 318. In someembodiments, CARs disclosed herein can comprise an extracellularligand-binding domain that specifically binds BCMA, CD8α human hinge andtransmembrane domains, the CD3ζ signaling domain, and 4-1BB signalingdomain.

Table 3 provides exemplary sequences of domains which can be used in theCARs disclosed herein.

TABLE 3 Exemplary sequences of CAR Components SEQ ID Domain Amino AcidSequence NO: CD8α signal peptide MALPVTALLLPLALLLHAARP 318FcγRIIIα hinge GLAVSTISSFFPPGYQ 319 CD8α hingeTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD 320 IgG1 hingeEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDV 321SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK CD8α IYIWAPLAGTCGVLLLSLVITLYC 322transmembrane (TM) domain 41BB intracellularKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 323 signaling domain (ISD)CD3ζ intracellular RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG 324signaling domain KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST (ISD)ATKDTYDALHMQALPPR FcεRI α-TM-IC (FcεRIFFIPLLVVILFAVDTGLFISTQQQVTFLLKIKRTRKGFRLLNPHPKPNPKNN 325 α chaintransmembrane and intracellular domain) FcεRIβ-ΔITAM (FcεRIMDTESNRRANLALPQEPSSVPAFEVLEISPQEVSSGRLLKSASSPPLHTWL 326 β chain withoutTVLKKEQEFLGVTQILTAMICLCFGTVVCSVLDISHIEGDIFSSFKAGYPFW ITAM)GAIFFSISGMLSIISERRNATYLVRGSLGANTASSIAGGTGITILIINLKKSLAYIHIHSCQKFFETKCFMASFSTEIVVMMLFLTILGLGSAVSLTICGAGEELKG NKVPE 41BB-IC (41BBco- KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 327 stimulatory domain)CD28-IC (CD28 co- RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 328stimulatory domain) FcεRIγ-SP (signal MIPAVVLLLLLLVEQAAA 329 peptide)FcεRI γ-ΔITAM (FcεRI LGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYEKS 330γ chain without ITAM) GSG-P2A (GSG-P2A GSGATNFSLLKQAGDVEENPGP 331ribosomal skip polypeptide) GSG-T2A (GSG-T2A GSGEGRGSLLTCGDVEENPGP 332ribosomal skip polypeptide)

Downregulation or mutation of target antigens is commonly observed incancer cells, creating antigen-loss escape variants. Thus, to offsettumor escape and render immune cell more specific to target, the BCMAspecific CAR can comprise one or more additional extracellularligand-binding domains, to simultaneously bind different elements intarget thereby augmenting immune cell activation and function. In oneembodiment, the extracellular ligand-binding domains can be placed intandem on the same transmembrane polypeptide, and optionally can beseparated by a linker. In some embodiments, said different extracellularligand-binding domains can be placed on different transmembranepolypeptides composing the CAR. In some embodiments, the inventionrelates to a population of CARs, each CAR comprising a differentextracellular ligand-binding domain. In a particular, the inventionrelates to a method of engineering immune cells comprising providing animmune cell and expressing at the surface of the cell a population ofCARs, each CAR comprising different extracellular ligand-bindingdomains. In another particular embodiment, the invention relates to amethod of engineering an immune cell comprising providing an immune celland introducing into the cell polynucleotides encoding polypeptidescomposing a population of CAR each one comprising differentextracellular ligand-binding domains. By population of CARs, it is meantat least two, three, four, five, six or more CARs each one comprisingdifferent extracellular ligand-binding domains. The differentextracellular ligand-binding domains according to the invention canpreferably simultaneously bind different elements in target therebyaugmenting immune cell activation and function. The invention alsorelates to an isolated immune cell which comprises a population of CARseach one comprising different extracellular ligand-binding domains.

In another aspect, the invention provides polynucleotides encoding anyof the CARs and polypeptides described herein. Polynucleotides can bemade and expressed by procedures known in the art.

In another aspect, the invention provides compositions (such as apharmaceutical compositions) comprising any of the cells of theinvention. In some embodiments, the composition comprises a cellcomprising a polynucleotide encoding any of the CARs described herein.In still other embodiments, the composition comprises either or both ofthe polynucleotides shown in SEQ ID NO: 367 and SEQ ID NO:368 below:

P5A Heavy Chain Variable Region

(SEQ ID NO: 367) GAGGTGCAGCTGCTGGAATCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCGCCATCAGCGATAGCGGCGGCAGCACCTACTACGCCGATAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCANATACTGGCCCATGGACATCTGGGGCCAGGGAACCTTGGTCACCGTCTCCTCA

P5A Light Chain Variable Region

(SEQ ID NO: 368) GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCAGGCGAAAGAGCCACCCTGTCCTGCAAAGCCAGCCAGAGCGTGTCCAGCAGCTACCTGGCCTGGTATCAGCAAAAGCCCGGCCAGGCTCCCCGGCTGCTGATGTACGATGCCAGCATCAGAGCCACCGGCATCCCCGACAGATTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGACTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACGGCAGCTGGCCCCTGACATTTGGCCAGGGCACAAAGGTGGAGATCAAA

In other embodiments, the composition comprises either or both of thepolynucleotides shown in SEQ ID NO: 369 and SEQ ID NO: 370 below:

P5AC1 Heavy Chain Variable Region

(SEQ ID NO: 369) GAGGTGCAGCTGCTGGAATCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGTAAAGGTTTGGAATGGGTTTCTGCTATTCTGTCGTCTGGTGGTTCTACTTACTATGCCGATTCTGTTAAGGGTAGATTCACCATTTCTAGAGACAACTCTAAGAACACCTTGTACTTGCAAATGAACTCCTTGAGAGCTGAAGATACTGCTGTTTATTACTGTGCTAGATACTGGCCAATGGATATTTGGGGTCAAGGTACTCTGGTCACCGTCTCCTCA

P5AC1 Light Chain Variable Region

(SEQ ID NO: 370) GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCTGGTGAAAGAGCTACTTTGTCTTGTAGAGGGGGTCAATCCGTTTCCTCTTCTTATTTGGCTTGGTATCAACAAAAACCAGGTCAAGCTCCAAGATTATTGATGTACGATGCTTCTATTAGAGCCACCGGTATTCCAGATAGATTTTCTGGTTCTGGTTCCGGTACTGATTTCACTTTGACTATCTCTAGATTGGAACCAGAAGATTTCGCTGTTTACTACTGTCAACAATATCAGTCTTGGCCATTGACTTTTGGTCAAGGTACAAAGGTTGAAATCAAA

In other embodiments, the composition comprises either or both of thepolynucleotides shown in SEQ ID NO: 371 and SEQ ID NO: 372 below:

PC1 Heavy Chain Variable Region

(SEQ ID NO: 371) GAGGTGCAGCTGCTGGAATCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACCCTATGAGCTGGGTGCGCCAGGCCCCTGGCAAAGGACTGGAATGGGTGTCCGCCATCGGAGGCTCTGGCGGCAGCACCTACTACGCCGATAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAAATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAGATACTGGCCCATGGACAGCTGGGGCCAGGGAACTTTGGTCACCGTCTCCTCA

PC1 Light Chain Variable Region

(SEQ ID NO: 372) GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCAGGCGAAAGAGCCACCCTGTCCTGCAAAGCCAGCCAGAGCGTGTCCAGCACATACCTGGCCTGGTATCAGCAAAAGCCCGGCCAGGCTCCCCGGCTGCTGATCTACGATGCCTCTTCTAGAGCCCCTGGCATCCCCGACAGATTCAGCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGACTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACAGCACCAGCCCCCTGACCTTTGGCCAGGGCACAAAGGTGGAGATCAAA.

In other embodiments, the composition comprises either or both of thepolynucleotides shown in SEQ ID NO: 373 and SEQ ID NO: 374 below:

PC1C12 Heavy Chain Variable Region

(SEQ ID NO: 373) GAGGTGCAGCTGCTGGAATCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACCCTATGAGCTGGGTGCGCCAGGCCCCTGGTAAAGGTTTGGAATGGGTTTCTGCTATTGGTGGTTCAGGTGGTTGGAGTTATTATGCCGATTCTGTTAAGGGTAGATTCACCATTTCTAGAGACAACTCTAAGAACACCTTGTACTTGCAAATGAACTCCTTGAGAGCTGAAGATACTGCTGTTTATTACTGTGCTAGATACTGGCCAATGGATTCTTGGGGTCAAGGTACTCTGGTCACCGTCTCCTCA

PC1C12 Light Chain Variable Region

(SEQ ID NO: 374) GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCTGGTGAAAGAGCTACTTTGTCTTGTTGGTTGTCTCAATCTGTTTCCTCTACTTACTTGGCTTGGTATCAACAAAAACCAGGTCAAGCTCCAAGATTATTGATCTACGATGCTTCTTCTAGAGCACCAGGTATTCCAGATAGATTTTCTGGTTCTGGTTCCGGTACTGATTTCACTTTGACTATCTCTAGATTGGAACCAGAAGATTTCGCTGTTTACTACTGCCAACAATACTCTGAGTGGCCATTGACTTTTGGTCAAGGTACAAAGGTTGAAATCAAA.

In other embodiments, the composition comprises either or both of thepolynucleotides shown in SEQ ID NO: 375 and SEQ ID NO: 376 below:

COM22 Heavy Chain Variable Region

(SEQ ID NO: 375) GAGGTGCAGCTGCTGGAATCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGTAAAGGTTTGGAATGGGTTTCTGCTATTTCTGATTCTGGTGGTTCTAGGTGGTATGCCGATTCTGTTAAGGGTAGATTCACCATTTCTAGAGACAACTCTAAGAACACCTTGTACTTGCAAATGAACTCCTTGAGAGCTGAAGATACTGCTGTTTATTACTGTACGCGGTACTGGCCAATGGATATTTGGGGTCAAGGTACTCTGGTCACCGTCTCCTCA

COM22 Light Chain Variable Region

(SEQ ID NO: 376) GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCTGGTGAAAGAGCTACTTTGTCTTGTTGGTTGTCTCAATCTGTTTCCTCTACTTACTTGGCTTGGTATCAACAAAAACCAGGTCAAGCTCCAAGATTATTGATCTACGATGCTTCTTCTAGAGCACCAGGTATTCCAGATAGATTTTCTGGTTCTGGTTCCGGTACTGATTTCACTTTGACTATCTCTAGATTGGAACCAGAAGATTTCGCTGTTTACTACTGCCAACAATACTCTGAGTGGCCATTGACTTTTGGTCAAGGTACAAAGGTTGAAATCAAA.

Expression vectors, and administration of polynucleotide compositionsare further described herein.

In another aspect, the invention provides a method of making any of thepolynucleotides described herein.

Polynucleotides complementary to any such sequences are also encompassedby the invention. Polynucleotides may be single-stranded (coding orantisense) or double-stranded, and may be DNA (genomic, cDNA orsynthetic) or RNA molecules. RNA molecules include HnRNA molecules,which contain introns and correspond to a DNA molecule in a one-to-onemanner, and mRNA molecules, which do not contain introns. Additionalcoding or non-coding sequences may, but need not, be present within apolynucleotide of the invention, and a polynucleotide may, but need not,be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes an antibody or a portion thereof) or may comprisea variant of such a sequence. Polynucleotide variants contain one ormore substitutions, additions, deletions and/or insertions such that theimmunoreactivity of the encoded polypeptide is not diminished, relativeto a native immunoreactive molecule. The effect on the immunoreactivityof the encoded polypeptide may generally be assessed as describedherein. Variants preferably exhibit at least about 70% identity, morepreferably, at least about 80% identity, yet more preferably, at leastabout 90% identity, and most preferably, at least about 95% identity toa polynucleotide sequence that encodes a native antibody or a portionthereof.

Two polynucleotide or polypeptide sequences are said to be “identical”if the sequence of nucleotides or amino acids in the two sequences isthe same when aligned for maximum correspondence as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, or 40 to about 50, in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M.O., 1978, A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M.O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; HeinJ., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E. W.and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971, Comb. Theor.11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath,P. H. A. and Sokal, R. R., 1973, Numerical Taxonomy the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA80:726-730.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e. the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

Variants may also, or alternatively, be substantially homologous to anative gene, or a portion or complement thereof. Such polynucleotidevariants are capable of hybridizing under moderately stringentconditions to a naturally occurring DNA sequence encoding a nativeantibody (or a complementary sequence).

Suitable “moderately stringent conditions” include prewashing in asolution of 5× SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C.-65° C., 5× SSC, overnight; followed by washing twice at 65° C. for 20minutes with each of 2×, 0.5× and 0.2× SSC containing 0.1% SDS.

As used herein, “highly stringent conditions” or “high stringencyconditions” are those that: (1) employ low ionic strength and hightemperature for washing, for example 0.015 M sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5× SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2× SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1× SSC containing EDTA at 55° C. The skilled artisanwill recognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Nonetheless, polynucleotides that vary due to differencesin codon usage are specifically contemplated by the invention. Further,alleles of the genes comprising the polynucleotide sequences providedherein are within the scope of the invention. Alleles are endogenousgenes that are altered as a result of one or more mutations, such asdeletions, additions and/or substitutions of nucleotides. The resultingmRNA and protein may, but need not, have an altered structure orfunction. Alleles may be identified using standard techniques (such ashybridization, amplification and/or database sequence comparison).

The polynucleotides of this invention can be obtained using chemicalsynthesis, recombinant methods, or PCR. Methods of chemicalpolynucleotide synthesis are well known in the art and need not bedescribed in detail herein. One of skill in the art can use thesequences provided herein and a commercial DNA synthesizer to produce adesired DNA sequence.

For preparing polynucleotides using recombinant methods, apolynucleotide comprising a desired sequence can be inserted into asuitable vector, and the vector in turn can be introduced into asuitable host cell for replication and amplification, as furtherdiscussed herein. Polynucleotides may be inserted into host cells by anymeans known in the art. Cells are transformed by introducing anexogenous polynucleotide by direct uptake, endocytosis, transfection,F-mating or electroporation. Once introduced, the exogenouspolynucleotide can be maintained within the cell as a non-integratedvector (such as a plasmid) or integrated into the host cell genome. Thepolynucleotide so amplified can be isolated from the host cell bymethods well known within the art. See, e.g., Sambrook et al., 1989.

Alternatively, PCR allows reproduction of DNA sequences. PCR technologyis well known in the art and is described in U.S. Pat. Nos. 4,683,195,4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase ChainReaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.

RNA can be obtained by using the isolated DNA in an appropriate vectorand inserting it into a suitable host cell. When the cell replicates andthe DNA is transcribed into RNA, the RNA can then be isolated usingmethods well known to those of skill in the art, as set forth inSambrook et al., 1989, supra, for example.

Suitable cloning vectors may be constructed according to standardtechniques, or may be selected from a large number of cloning vectorsavailable in the art. While the cloning vector selected may varyaccording to the host cell intended to be used, useful cloning vectorswill generally have the ability to self-replicate, may possess a singletarget for a particular restriction endonuclease, and/or may carry genesfor a marker that can be used in selecting clones containing the vector.Suitable examples include plasmids and bacterial viruses, e.g., pUC18,pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19,pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such aspSA3 and pAT28. These and many other cloning vectors are available fromcommercial vendors such as BioRad, Strategene, and Invitrogen.

Expression vectors generally are replicable polynucleotide constructsthat contain a polynucleotide according to the invention. It is impliedthat an expression vector must be replicable in the host cells either asepisomes or as an integral part of the chromosomal DNA. Suitableexpression vectors include but are not limited to plasmids, viralvectors, including adenoviruses, adeno-associated viruses, retroviruses,cosmids, and expression vector(s) disclosed in PCT Publication No. WO87/04462. Vector components may generally include, but are not limitedto, one or more of the following: a signal sequence; an origin ofreplication; one or more marker genes; suitable transcriptionalcontrolling elements (such as promoters, enhancers and terminator). Forexpression (i.e., translation), one or more translational controllingelements are also usually required, such as ribosome binding sites,translation initiation sites, and stop codons.

The vectors containing the polynucleotides of interest can be introducedinto the host cell by any of a number of appropriate means, includingelectroporation, transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (e.g., where thevector is an infectious agent such as vaccinia virus). The choice ofintroducing vectors or polynucleotides will often depend on features ofthe host cell.

A polynucleotide encoding a BCMA specific CAR disclosed herein may existin an expression cassette or expression vector (e.g., a plasmid forintroduction into a bacterial host cell, or a viral vector such as abaculovirus vector for transfection of an insect host cell, or a plasmidor viral vector such as a lentivirus for transfection of a mammalianhost cell). In some embodiments, a polynucleotide or vector can includea nucleic acid sequence encoding ribosomal skip sequences such as, forexample without limitation, a sequence encoding a 2A peptide. 2Apeptides, which were identified in the Aphthovirus subgroup ofpicornaviruses, causes a ribosomal “skip” from one codon to the nextwithout the formation of a peptide bond between the two amino acidsencoded by the codons (see (Donnelly and Elliott 2001; Atkins, Wills etal. 2007; Doronina, Wu et al. 2008)). By “codon” is meant threenucleotides on an mRNA (or on the sense strand of a DNA molecule) thatare translated by a ribosome into one amino acid residue. Thus, twopolypeptides can be synthesized from a single, contiguous open readingframe within an imRNA when the polypeptides are separated by a 2Aoligopeptide sequence that is in frame. Such ribosomal skip mechanismsare well known in the art and are known to be used by several vectorsfor the expression of several proteins encoded by a single messengerRNA.

To direct transmembrane polypeptides into the secretory pathway of ahost cell, in some embodiments, a secretory signal sequence (also knownas a leader sequence, prepro sequence or pre sequence) is provided in apolynucleotide sequence or vector sequence. The secretory signalsequence is operably linked to the transmembrane nucleic acid sequence,i.e., the two sequences are joined in the correct reading frame andpositioned to direct the newly synthesized polypeptide into thesecretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the nucleic acid sequence encoding thepolypeptide of interest, although certain secretory signal sequences maybe positioned elsewhere in the nucleic acid sequence of interest (see,e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat.No. 5,143,830). In some embodiments the signal peptide comprises theamino acid sequence shown in SEQ ID NO: 318 or 329. Those skilled in theart will recognize that, in view of the degeneracy of the genetic code,considerable sequence variation is possible among these polynucleotidemolecules. In some embodiments, nucleic acid sequences of the inventionare codon-optimized for expression in mammalian cells, preferably forexpression in human cells. Codon-optimization refers to the exchange ina sequence of interest of codons that are generally rare in highlyexpressed genes of a given species by codons that are generally frequentin highly expressed genes of such species, such codons encoding theamino acids as the codons that are being exchanged.

In some embodiments, a polynucleotide according to the inventioncomprises the nucleic acid sequence selected from the group consistingof: SEQ. ID NO: 1397. The invention relates to polynucleotidescomprising a nucleic acid sequence that has at least 70%, preferably atleast 80%, more preferably at least 90%, 95%, 97%, or 99% sequenceidentity with nucleic acid sequence selected from the group consistingof SEQ ID NO: 1397.

Methods of Engineering an Immune Cell

Methods of preparing immune cells for use in immunotherapy are providedherein. In some embodiments, the methods comprise introducing a CARaccording to the invention into immune cells, and expanding the cells.In some embodiments, the invention relates to a method of engineering animmune cell comprising: providing a cell and expressing at the surfaceof the cell at least one CAR as described above. Methods for engineeringimmune cells are described in, for example, PCT Patent ApplicationPublication Nos. WO/2014/039523, WO/2014/184741, WO/2014/191128,WO/2014/184744, and WO/2014/184143, each of which is incorporated hereinby reference in its entirety. In some embodiments, the method comprises:transfecting the cell with at least one polynucleotide encoding CAR asdescribed above, and expressing the polynucleotides in the cell.

In some embodiments, the polynucleotides are present in lentiviralvectors for stable expression in the cells.

In some embodiments, the method can further comprise a step ofgenetically modifying a cell by inactivating at least one geneexpressing, for example without limitation, a component of the TCR, atarget for an immunosuppressive agent, an HLA gene, and/or an immunecheckpoint protein such as, for example, PDCD1 or CTLA-4. Byinactivating a gene it is intended that the gene of interest is notexpressed in a functional protein form. In some embodiments, the gene tobe inactivated is selected from the group consisting of, for examplewithout limitation, TCRα, TCRβ, CD52, GR, PD-1, and CTLA-4. In someembodiments the method comprises inactivating one or more genes byintroducing into the cells a rare-cutting endonuclease able toselectively inactivate a gene by selective DNA cleavage. In someembodiments the rare-cutting endonuclease can be, for example, atranscription activator-like effector nuclease (TALE-nuclease) or Cas9endonuclease.

In some embodiments, an additional catalytic domain is used with arare-cutting endonuclease to enhance its capacity to inactivate targetedgenes. For example, an additional catalytic domain can be a DNAend-processing enzyme. Non-limiting examples of DNA end-processingenzymes include 5-3′ exonucleases, 3-5′ exonucleases, 5-3′ alkalineexonucleases, 5′ flap endonucleases, helicases, hosphatase, hydrolasesand template-independent DNA polymerases. Non-limiting examples of suchcatalytic domain comprise of a protein domain or catalytically activederivate of the protein domain selected from the group consisting ofhExol (EXO1_HUMAN), Yeast Exol (EXO1_YEAST), E. coli Exol, Human TREX2,Mouse TREX1, Human TREX1, Bovine TREX1, Rat TREX1, TdT (terminaldeoxynucleotidyl transferase) Human DNA2, Yeast DNA2 (DNA2_YEAST). Insome embodiments, an additional catalytic domain can have a3′-5′-exonuclease activity, and In some embodiments, said additionalcatalytic domain is TREX, more preferably TREX2 catalytic domain(WO2012/058458). In some embodiments, said catalytic domain is encodedby a single chain TREX polypeptide. The additional catalytic domain maybe fused to a nuclease fusion protein or chimeric protein. In someembodiments, the additional catalytic domain is fused using, forexample, a peptide linker.

In some embodiments, the method further comprises a step of introducinginto cells an exogeneous nucleic acid comprising at least a sequencehomologous to a portion of the target nucleic acid sequence, such thathomologous recombination occurs between the target nucleic acid sequenceand the exogeneous nucleic acid. In some embodimentss, said exogenousnucleic acid comprises first and second portions which are homologous toregion 5′ and 3′ of the target nucleic acid sequence, respectively. Theexogenous nucleic acid may also comprise a third portion positionedbetween the first and the second portion which comprises no homologywith the regions 5′ and 3′ of the target nucleic acid sequence.Following cleavage of the target nucleic acid sequence, a homologousrecombination event is stimulated between the target nucleic acidsequence and the exogenous nucleic acid. In some embodiments, homologoussequences of at least about 50 bp, greater than about 100 bp, or greaterthan about 200 bp can be used within the donor matrix. The exogenousnucleic acid can be, for example without limitation, from about 200 bpto about 6000 bp, more preferably from about 1000 bp to about 2000 bp.Shared nucleic acid homologies are located in regions flanking upstreamand downstream the site of the break, and the nucleic acid sequence tobe introduced is located between the two arms.

In some embodiments, a nucleic acid successively comprises a firstregion of homology to sequences upstream of said cleavage; a sequence toinactivate a targeted gene selected from the group consisting of TCRα,TCRβ3, CD52, glucocorticoid receptor (GR), deoxycytidine kinase (DCK),and an immune checkpoint protein such as for example programmed death-1(PD-1); and a second region of homology to sequences downstream of thecleavage. The polynucleotide introduction step can be simultaneous,before or after the introduction or expression of the rare-cuttingendonuclease. Depending on the location of the target nucleic acidsequence wherein break event has occurred, such exogenous nucleic acidcan be used to knock-out a gene, e.g. when exogenous nucleic acid islocated within the open reading frame of the gene, or to introduce newsequences or genes of interest. Sequence insertions by using suchexogenous nucleic acid can be used to modify a targeted existing gene,by correction or replacement of the gene (allele swap as a non-limitingexample), or to up- or down-regulate the expression of the targeted gene(promoter swap as non-limiting example), the targeted gene correction orreplacement. In some embodiments, inactivation of a genes selected fromthe group consisting of TCRα, TCRβ, CD52, GR, DCK, and immune checkpointproteins, can be done at a precise genomic location targeted by aspecific TALE-nuclease, wherein said specific TALE-nuclease catalyzes acleavage and wherein the exogenous nucleic acid successively comprisingat least a region of homology and a sequence to inactivate one targetedgene selected from the group consisting of TCRα, TCRβ, CD52, GR, DCK,immune checkpoint proteins which is integrated by homologousrecombination. In some embodiments, several genes can be, successivelyor at the same time, inactivated by using several TALE-nucleasesrespectively and specifically targeting one defined gene and severalspecific polynucleotides for specific gene inactivation.

In some embodiments, the method comprises inactivation of one or moreadditional genes selected from the group consisting of TCRα, TCRβ, CD52,GR, DCK, and immune checkpoint proteins. In some embodiments,inactivation of a gene can be accomplished by introducing into the cellsat least one rare-cutting endonuclease such that the rare-cuttingendonuclease specifically catalyzes cleavage in a targeted sequence ofthe cell genome; and optionally, introducing into the cells an exogenousnucleic acid successively comprising a first region of homology tosequences upstream of the cleavage, a sequence to be inserted in thegenome of the cell, and a second region of homology to sequencesdownstream of the cleavage; wherein the introduced exogenous nucleicacid inactivates a gene and integrates at least one exogenouspolynucleotide sequence encoding at least one recombinant protein ofinterest. In some embodiments, theexogenous polynucleotide sequence isintegrated within a gene encoding a protein selected from the groupconsisting of TCRα, TCRβ, CD52, GR, DCK, and immune checkpoint protein.

In another aspect, a step of genetically modifying cells cancomprise:modifying T cells by inactivating at least one gene expressinga target for an immunosuppressive agent, and; expanding the cells,optionally in presence of the immunosuppressive agent. Animmunosuppressive agent is an agent that suppresses immune function byone of several mechanisms of action. An immunosuppressive agent candiminish the extent and/or voracity of an immune response. Non-limitingexamples of immunosuppressive agents iinclude calcineurin inhibitors,targets of rapamycin, interleukin-2 α-chain blockers, inhibitors ofinosine monophosphate dehydrogenase, inhibitors of dihydrofolic acidreductase, corticosteroids, and immunosuppressive antimetabolites. Somecytotoxic immunosuppressants act by inhibiting DNA synthesis. Others mayact through activation of T cells or by inhibiting the activation ofhelper cells. The methods according to the invention allow conferringimmunosuppressive resistance to T cells for immunotherapy byinactivating the target of the immunosuppressive agent in T cells. Asnon-limiting examples, targets for immunosuppressive agent can be areceptor for an immunosuppressive agent such as for example withoutlimtiation CD52, glucocorticoid receptor (GR), FKBP family gene members,and cyclophilin family gene members.

In some embodiments, the genetic modification of the method invovlesexpression, in provided cells to engineer, of one rare-cuttingendonuclease such that the rare-cutting endonuclease specificallycatalyzes cleavage in one targeted gene, thereby inactivating thetargeted gene. In some embodiments, a method of engineering cellscomprises at least one of the following steps: providing a T cell, suchas from a cell culture or from a blood sample; selecting a gene in the Tcell expressing a target for an immunosuppressive agent; introducinginto the T cell a rare-cutting endonuclease able to selectivelyinactivate by DNA cleavage, preferably by double-strand break the geneencoding a target for the immunosuppressive agent, and expanding thecells, optionally in presence of the immunosuppressive agent.

In some embodiments, the method comprises: providing a T cell, such asfrom a cell culture or from a blood sample; selecting a gene in the Tcell wherein the gene expresses a target for an immunosuppressive agent;transfecting the T cell with nucleic acid encoding a rare-cuttingendonuclease able to selectively inactivate by DNA cleavage, preferablyby double-strand break the gene encoding a target for theimmunosuppressive agent, and expressing the rare-cutting endonucleasesinto the T cells; and expanding the cells, optionally in presence of theimmunosuppressive agent.

In some embodiments, the rare-cutting endonuclease specifically targetsCD52 or GR. In some embodiments, the gene selected for inactivationencodes CD52, and the immunosuppressive treatment comprises a humanizedantibody targeting CD52 antigen. In some embodiments, the gene selectedfor inactivation encodes GR, and the immunosuppressive treatmentcomprises a corticosteroid such as dexamethasone. In some embodiments,the gene selected for inactivation is a FKBP family gene member or avariant thereof and the immunosuppressive treatment comprises FK506,also known as Tacrolimus or fujimycin. In some embodiments, the FKBPfamily gene member is FKBP12 or a variant thereof. In some embodiments,gene selected for inactivation is a cyclophilin family gene member or avariant thereof and the immunosuppressive treatment comprisescyclosporine.

In some embodiments, the rare-cutting endonuclease can be, for example,a meganuclease, a zinc finger nuclease, or a TALE-nuclease (TALEN). Insome embodiments, the rare-cutting endonuclease is a TALE-nuclease.

Also provided herein are methods of engineering T cells, suitable forimmunotherapy, wherein the methods comprise: genetically modifying Tcells by inactivating at least immune checkpoint protein. In someembodiments the immune checkpoint protein is, for example, PD-1 and/orCTLA-4.In some embodiments, methods of genetically modifying a cellcomprises: modifying T cells by inactivating at least one immunecheckpoint protein; and expanding the cells. Immune checkpoint proteinsinclude, but are not limited to Programmed Death 1 (PD-1, also known asPDCD1 or CD279, accession number: NM_005018), Cytotoxic T-LymphocyteAntigen 4 (CTLA-4, also known as CD152, GenBank accession numberAF414120.1), LAG3 (also known as CD223, accession number: NM_002286.5),Tim3 (also known as HAVCR2, GenBank accession number: JX049979.1), BTLA(also known as CD272, accession number: NM_181780.3), BY55 (also knownas CD160, GenBank accession number: CR541888.1), TIGIT (also known asVSTM3, accession number: NM_173799), B7H5 (also known as C10orf54,homolog of mouse vista gene, accession number: NM_022153.1), LAIR1 (alsoknown as CD305, GenBank accession number: CR542051.1), SIGLEC10(GeneBank accession number: AY358337.1), 2B4 (also known as CD244,accession number: NM_001166664.1), which directly inhibit immune cells.For example, CTLA-4 is a cell-surface protein expressed on certain CD4and CD8 T cells; when engaged by its ligands (B7-1 and B7-2) on antigenpresenting cells, T cell activation and effector function are inhibited.

In some embodiments, said method to engineer cells comprises at leastone of the following steps: providing a T cell, such as from a cellculture or from a blood sample; introducing into the T cell arare-cutting endonuclease able to selectively inactivate by DNAcleavage, preferably by double-strand break one gene encoding a immunecheckpoint protein; and expanding the cells. In some embodiments, themethod comprises: providing a T cell, such as from a cell culture orfrom a blood sample; transfecting said T cell with nucleic acid encodinga rare-cutting endonuclease able to selectively inactivate by DNAcleavage, preferably by double-strand break a gene encoding a immunecheckpoint protein; expressing the rare-cutting endonucleases into the Tcells; expanding the cells. In some embodiments, the rare-cuttingendonuclease specifically targets a gene selected from the groupconsisting of: PD-1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1,SIGLEC10, 2B4, TCRα, and TCRβ. In some embodiments, the rare-cuttingendonuclease can be a meganuclease, a zinc finger nuclease or aTALE-nuclease. In some embodiments, the rare-cutting endonuclease is aTALE-nuclease.

In some embodiments, the present invention can be particularly suitablefor allogeneic immunotherapy. In such embodiments, cells may be modifiedby a method comprising: inactivating at least one gene encoding acomponent of the T cell receptor (TCR) in T cells; and expanding the Tcells. In some embodiments, the genetic modification of the methodrelies on the expression, in provided cells to engineer, of onerare-cutting endonuclease such that the rare-cutting endonucleasespecifically catalyzes cleavage in one targeted gene therebyinactivating the targeted gene. In some embodiments, said method toengineer cells comprises at least one of the following steps: providinga T cell, such as from a cell culture or from a blood sample;introducing into the T cell a rare-cutting endonuclease able toselectively inactivate by DNA cleavage, preferably by double-strandbreak at least one gene encoding a component of the T cell receptor(TCR), and expanding the cells.

In some embodiments, the method comprises: providing a T cell, such asfrom a cell culture or from a blood sample; transfecting said T cellwith nucleic acid encoding a rare-cutting endonuclease able toselectively inactivate by DNA cleavage, preferably by double-strandbreak at least one gene encoding a component of the T cell receptor(TCR); expressing the rare-cutting endonucleases into the T cells;sorting the transformed T cells, which do not express TCR on their cellsurface;and expanding the cells.

In some embodiments, the rare-cutting endonuclease can be ameganuclease, a zinc finger nucleasec or a TALE-nuclease. In someembodiments, the rare-cutting endonuclease is a TALE-nuclease. In someembodiments the TALE-nucleases recognize and cleave a sequence encodingTCRα or TCRβ. In some embodiments a TALE-nuclease comprises apolypeptide sequence selected from the amino acid sequence shown in SEQID NO: 334, 335, 336, 337, 338, 339, 340, or 341

TALE-nuclease polypeptide sequences: Repeat TRAC_T01-L (SEQ ID NO: 334)LTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE Repeat TRAC_T01-R (SEQ ID NO: 335)LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE Repeat TRBC_T01-L (SEQ ID NO: 336)LTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGRPALE Repeat TRBC_T01-R (SEQ ID NO: 337)NPQRSTVVVYLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE Repeat TRBC_T02-L (SEQ ID NO:338) LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE Repeat TRBC_T02-R (SEQ ID NO: 339)LTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE Repeat CD52_T02-L (SEQ ID NO: 340)LTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGRPALE Repeat CD52_T02-R (SEQ ID NO: 341)LTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE

In another aspect, one another step of genetically modifying cell can bea method of expanding TCRα deficient T cells comprising introducing intothe T cell pTα (also known as preTCRα) or a functional variant thereofand expanding the cells, optionally through stimulation of the CD3complex. In some embodiments, the method comprises: a) transfecting thecells with nucleic acid encoding at least a fragment of pTα to supportCD3 surface expression; b) expressing said pTα into the cells; and c)expanding the cells, optionally through stimulation of the CD3 complex.

Also provided are methods of preparing T cells for immunotherapycomprising steps of the method for expansion for T cell. In someembodiments, the pTα polynucleotide sequence can be introduced randomlyor by homologous recombination. In some embodiments, the insertion canbe associated with the inactivation of the TCRα gene. Differentfunctional variants of pTα can be used. A “functional variant” of thepeptide refers to a molecule substantially similar to either the entirepeptide or a fragment thereof. A “fragment” of the pTα or functionalvariant thereof refers to any subset of the molecule, that is, a shorterpeptide than the full-length pTα. In some embodiments, pTα or functionalvariants can be, for example, full-length pTα or a C-terminal truncatedpTα version. C-terminal truncated pTα lacks in C-terminal end one ormore residues. As non limiting examples, C-terminal truncated pTαversion lacks 18, 48, 62, 78, 92, 110 or 114 residues from theC-terminus of the protein. Amino acid sequence variants of the peptidecan be prepared by mutations in the DNA which encodes the peptide. Suchfunctional variants include, for example, deletions from, or insertionsor substitutions of, residues within the amino acid sequence. Anycombination of deletion, insertion, and substitution may also be made toarrive at the final construct, provided that the final constructpossesses the desired activity, in particular the restoration of afunctional CD3 complex. In preferred embodiment, at least one mutationis introduced in the different pTα versions as described above to affectdimerization. As non limiting example, mutated residue can be at leastW46R, D22A, K24A, R102A or R117A of the human pTα protein or alignedpositions using CLUSTALW method on pTα family or homologue member.Preferably pTα or variant thereof as described above comprise themutated residue W46R or the mutated residues D22A, K24A, R102A andR117A. In some embodiments, said pTα or variants are also fused to asignal-transducing domain such as CD28, OX40, ICOS, CD27, CD137 (4-1BB)and CD8 as non limiting examples. The extracellular domain of pTα orvariants as described above can be fused to a fragment of the TCRαprotein, particularly the transmembrane and intracellular domain ofTCRα. pTα variants can also be fused to the intracellular domain ofTCRα.

In some embodiments, pTα versions can be fused to an extracellularligand-binding domain. In some embodiments, pTα or functional variantthereof is fused to a single chain antibody fragment (scFv) comprisingthe light and the heavy variable fragment of a target antigen specificmonoclonal antibody joined by a flexible linker.

The term “TCRα deficient T cell” refers to an isolated T cell that lacksexpression of a functional TCRα chain. This may be accomplished bydifferent means, as non limiting examples, by engineering a T cell suchthat it does not express any functional TCRα on its cell surface or byengineering a T cell such that it produces very little functional TCRαchain on its surface or by engineering a T cell to express mutated ortruncated form of TCRα chain. TCRα deficient cells can no longer beexpanded through CD3 complex. Thus, to overcome this problem and toallow proliferation of TCRα deficient cells, pTα or functional variantthereof is introduced into the cells, thus restoring a functional CD3complex. In some embodiments, the method further comprises introducinginto said T cells rare-cutting endonucleases able to selectivelyinactivate by DNA cleavage one gene encoding one component of the T cellreceptor (TCR). In some embodiments, the rare-cutting endonuclease is aTALE-nuclease.

In another aspect, engineered T cells obtained by the methods describedherein can be contacted with bispecific antibodies. For example, the Tcells can be contacted with bispecific antibodies ex vivo prior toadministration to a patient, or in vivo following administration to apatient. Bispecific antibodies comprise two variable regions withdistinct antigen properties that facilitate bringing the engineeredcells into proximity to a target antigen. As a non-limiting example, abispecific antibody can be directed against a tumor marker andlymphocyte antigen, such as for example without limitation CD3, and hasthe potential to redirect and activate any circulating T cells againsttumors.

In some embodiments, polynucleotides encoding polypeptides according tothe present invention can be mRNA which is introduced directly into thecells, for example by electroporation. In some embodiments, cytoPulsetechnology can be used to transiently permeabilize living cells fordelivery of material into the cells. Parameters can be modified in orderto determine conditions for high transfection efficiency with minimalmortality.

Also provided herein are methods of transfecting T cell. In someembodiments, the method comprises: contacting a T cell with RNA andapplying to T cell an agile pulse sequence consisting of: (a) anelectrical pulse with a voltage range from about 2250 to 3000 V percentimeter; (b) a pulse width of 0.1 ms; (c) a pulse interval of about0.2 to 10 ms between the electrical pulses of step (a) and (b); (d) anelectrical pulse with a voltage range from about 2250 to 3000 V with apulse width of about 100 ms and a pulse interval of about 100 ms betweenthe electrical pulse of step (b) and the first electrical pulse of step(c); and (e) four electrical pulses with a voltage of about 325 V with apulse width of about 0.2 ms and a pulse interval of 2 ms between each of4 electrical pulses. In some embodiments, a method of transfecting Tcell comprising contacting said T cell with RNA and applying to T cellan agile pulse sequence comprising: (a) an electrical pulse with avoltage of about 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450,2500, 2600, 2700, 2800, 2900 or 3000V per centimeter; (b) a pulse widthof 0.1 ms; (c) and a pulse interval of about 0.2, 0.5, 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 ms between the electrical pulses of step (a) and (b); (d)one electrical pulse with a voltage range from about 2250, of 2250,2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800,2900 or 3000V with a pulse width of 100 ms and a pulse interval of 100ms between the electrical pulse of step (b) and the first electricalpulse of step (c); and (e) 4 electrical pulses with a voltage of about325 V with a pulse width of about 0.2 ms and a pulse interval of about 2ms between each of 4 electrical pulses. Any values included in the valuerange described above are disclosed in the present application.Electroporation medium can be any suitable medium known in the art. Insome embodiments, the electroporation medium has conductivity in a rangespanning about 0.01 to about 1.0 milliSiemens.

In some embodiments, as non limiting examples, an RNA encodes arare-cutting endonuclase, one monomer of the rare-cutting endonucleasesuch as half-TALE-nuclease, a CAR, at least one component of themulti-chain chimeric antigen receptor, a pTα or functional variantthereof, an exogenous nucleic acid, and/or one additional catalyticdomain.

Engineered Immune Cells

The invention also provides engineered immune cells comprising any ofthe CAR polynucleotides described herein. In some embodiments, a CAR canbe introduced into an immune cell as a transgene via a plasmid vector.In some embodiments, the plasmid vector can also contain, for example, aselection marker which provides for identification and/or selection ofcells which received the vector.

CAR polypeptides may be synthesized in situ in the cell afterintroduction of polynucleotides encoding the CAR polypeptides into thecell. Alternatively, CAR polypeptides may be be produced outside ofcells, and then introduced into cells. Methods for introducing apolynucleotide construct into cells are known in the art. In someembodiments, stable transformation methods can be used to integrate thepolynucleotide construct into the genome of the cell. In otherembodiments, transient transformation methods can be used to transientlyexpress the polynucleotide construct, and the polynucleotide constructnot integrated into the genome of the cell. In other embodiments,virus-mediated methods can be used. The polynucleotides may beintroduced into a cell by any suitable means such as for example,recombinant viral vectors (e.g. retroviruses, adenoviruses), liposomes,and the like. Transient transformation methods include, for examplewithout limitation, microinjection, electroporation or pa rticlebombardment. Polynucleotides may be included in vectors, such as forexample plasmid vectors or viral vectors.

Also provided herein are isolated cells and cell lines obtained by theabove-described methods of engineering cells provided herein. In someembodiments, an isolated cell comprises at least one CAR as describedabove. In some embodiments, an isolated cell comprises a population ofCARs, each CAR comprising different extracellular ligand-bindingdomains.

Also provided herein are isolated immune cells obtained according to anyone of the methods described above. Any immune cell capable ofexpressing heterologous DNAs can be used for the purpose of expressingthe CAR of interest. In some embodiments, the immune cell is a T cell.In some embodiments, an immune cell can be derived from, for examplewithout limitation, a stem cell. The stem cells can be adult stem cells,non-human embryonic stem cells, more particularly non-human stem cells,cord blood stem cells, progenitor cells, bone marrow stem cells, inducedpluripotent stem cells, totipotent stem cells or hematopoietic stemcells. Representative human cells are CD34+ cells. The isolated cell canalso be a dendritic cell, killer dendritic cell, a mast cell, a NK-cell,a B-cell or a T cell selected from the group consisting of inflammatoryT-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes orhelper T-lymphocytes. I n some embodiments, the cell can be derived fromthe group consisting of CD4+ T-lymphocytes and CD8+ T-lymphocytes.

Prior to expansion and genetic modification, a source of cells can beobtained from a subject through a variety of non-limiting methods. Cellscan be obtained from a number of non-limiting sources, includingperipheral blood mononuclear cells, bone marrow, lymph node tissue, cordblood, thymus tissue, tissue from a site of infection, ascites, pleuraleffusion, spleen tissue, and tumors. In some embodiments, any number ofT cell lines available and known to those skilled in the art, may beused. In some embodiments, cells can be derived from a healthy donor,from a patient diagnosed with cancer or from a patient diagnosed with aninfection. In some embodiments, cells can be part of a mixed populationof cells which present different phenotypic characteristics.

Also provided herein are cell lines obtained from a transformed T cellaccording to any of the above-described methods. Also provided hereinare modified cells resistant to an immunosuppressive treatment. In someembodiments, an isolated cell according to the invention comprises apolynucleotide encoding a CAR.

The immune cells of the invention can be activated and expanded, eitherprior to or after genetic modification of the T cells, using methods asgenerally described, for example without limitation, in U.S. Pat. Now.6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466;6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843;5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent ApplicationPublication No. 20060121005. T cells can be expanded in vitro or invivo. Generally, the T cells of the invention can be expanded, forexample, by contact with an agent that stimulates a CD3 TCR complex anda co-stimulatory molecule on the surface of the T cells to create anactivation signal for the T cell. For example, chemicals such as calciumionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogeniclectins like phytohemagglutinin (PHA) can be used to create anactivation signal for the T cell.

In some embodiments, T cell populations may be stimulated in vitro bycontact with, for example, an anti-CD3 antibody, or antigen-bindingfragment thereof, or an anti-CD2 antibody immobilized on a surface, orby contact with a protein kinase C activator (e.g., bryostatin) inconjunction with a calcium ionophore. For co-stimulation of an accessorymolecule on the surface of the T cells, a ligand that binds theaccessory molecule is used. For example, a population of T cells can becontacted with an anti-CD3 antibody and an anti-CD28 antibody, underconditions appropriate for stimulating proliferation of the T cells.Conditions appropriate for T cell culture include an appropriate media(e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 5, (Lonza))that may contain factors necessary for proliferation and viability,including serum (e.g., fetal bovine or human serum), interleukin-2(IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGFp,and TNF, or any other additives for the growth of cells known to theskilled artisan. Other additives for the growth of cells include, butare not limited to, surfactant, plasmanate, and reducing agents such asN-acetyl-cysteine and 2-mercaptoethanoi. Media can include RPMI 1640,A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1, and X-Vivo 20, Optimizer, withadded amino acids, sodium pyruvate, and vitamins, either serum-free orsupplemented with an appropriate amount of serum (or plasma) or adefined set of hormones, and/or an amount of cytokine(s) sufficient forthe growth and expansion of T cells. Antibiotics, e.g., penicillin andstreptomycin, are included only in experimental cultures, not incultures of cells that are to be infused into a subject. The targetcells are maintained under conditions necessary to support growth, forexample, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g.,air plus 5% CO₂). T cells that have been exposed to varied stimulationtimes may exhibit different characteristics

In some embodiments, the cells of the invention can be expanded byco-culturing with tissue or cells. The cells can also be expanded invivo, for example in the subject's blood after administrating the cellinto the subject.

In some embodiments, an isolated cell according to the present inventioncomprises one inactivated gene selected from the group consisting ofCD52, GR, PD-1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1,SIGLEC10, 2B4, HLA, TCRα and TCRβ and/or expresses a CAR, a multi-chainCAR and/or a pTα transgene. In some embodiments, an isolated cellcomprises polynucleotides encoding polypeptides comprising a multi-chainCAR. In some embodiments, the isolated cell according to the presentinvention comprises two inactivated genes selected from the groupconsisting of: CD52 and GR, CD52 and TCRα, CDR52 and TCRβ, GR and TCRα,GR and TCRβ, TCRα and TCRβ, PD-1 and TCRα, PD-1 and TCRβ, CTLA-4 andTCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, Tim3 and TCRα, Tim3and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ,TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 andTCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 andTCRα, 2B4 and TCRβ and/or expresses a CAR, a multi-chain CAR and a pTαtransgene.

In some embodiments, TCR is rendered not functional in the cellsaccording to the invention by inactivating TCRα gene and/or TCRβgene(s). In some embodiments, a method to obtain modified cells derivedfrom an individual is provided, wherein the cells can proliferateindependently of the major histocompatibility complex (MHC) signalingpathway. Modified cells, which can proliferate independently of the MHCsignaling pathway, susceptible to be obtained by this method areencompassed in the scope of the present invention. Modified cellsdisclosed herein can be used in for treating patients in need thereofagainst Host versus Graft (HvG) rejection and Graft versus Host Disease(GvHD); therefore in the scope of the present invention is a method oftreating patients in need thereof against Host versus Graft (HvG)rejection and Graft versus Host Disease (GvHD) comprising treating saidpatient by administering to said patient an effective amount of modifiedcells comprising inactivated TCRα and/or TCRβ genes.

In some embodiments, the immune cells are engineered to be resistant toone or more chemotherapy drugs. The chemotherapy drug can be, forexample, a purine nucleotide analogue (PNA), thus making the immune cellsuitable for cancer treatment combining adoptive immunotherapy andchemotherapy. Exemplary PNAs include, for example, clofarabine,fludarabine, and cytarabine, alone or in combination. PNAs aremetabolized by deoxycytidine kinase (dCK) into mono-, di-, andtri-phosphate PNA. Their tri-phosphate forms compete with ATP for DNAsynthesis, act as pro-apoptotic agents, and are potent inhibitors ofribonucleotide reductase (RNR), which is involved in trinucleotideproduction. Provided herein are BCMA specific CAR-T cells comprising aninactivated dCK gene. In some embodiments, the dCK knockout cells aremade by transfection of T cells using polynucleotides encoding specificTAL-nulcease directed against dCK genes by, for example, electroporationof mRNA. The dCK knockout BCMA specific CAR-T cells are resistant toPNAs, including for example clorofarabine and/or fludarabine, andmaintain T cell cytotoxic activity toward BCMA-expressing cells.

In some embodiments, isolated cells or cell lines of the invention cancomprise a pTα or a functional variant thereof. In some embodiments, anisolated cell or cell line can be further genetically modified byinactivating the TCRα gene.

In some embodiments, the CAR-T cell comprises a polynucleotide encodinga suicide polypeptide, such as for example RQR8. See, e.g.,WO2013153391A, which is hereby incorporated by reference in itsentirety. In CAR-T cells comprising the polynucleotide, the suicidepolypeptide is expressed at the surface of a CAR-T cell. In someembodiments, the suicide polypeptide comprises the amino acid sequenceshown in SEQ ID NO: 342.

(SEQ ID NO: 342) CPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVThe suicide polypeptide may also comprise a signal peptide at the aminoterminus. In some embodiments, the suicide polypeptide comprises theamino acid sequence shown in SEQ ID NO: 400.

(SEQ ID NO: 400) MGTSLLCWMALCLLGADHADACPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVC KCPRPVVWhen the suicide polypeptide is expressed at the surface of a CAR-Tcell, binding of rituximab to the R epitopes of the polypeptide causeslysis of the cell. More than one molecule of rituximab may bind perpolypeptide expressed at the cell surface. Each R epitope of thepolypeptide may bind a separate molecule of rituximab. Deletion of BCMAspecific CAR-T cells may occur in vivo, for example by administeringrituximab to a patient. The decision to delete the transferred cells mayarise from undesirable effects being detected in the patient which areattributable to the transferred cells, such as for example, whenunacceptable levels of toxicity are detected.

In some embodiments, the CAR-T cell comprises a selected epitope withinthe scFv having a specificity to be recognized by a specific antibody.See, e.g., PCT application “mAb-DRIVEN CHIMERIC ANTIGEN RECEPTOR SYSTEMSFOR SORTING/DEPLETING ENGINEERED IMMUNE CELLS,” filed on Jan. 25, 2016,which is hereby incorporated by reference in its entirety. Such anepitope facilitates sorting and/or depleting the CAR-T cells. Theepitope can be selected from any number of epitopes known in the art. Insome embodiments, the epitope can be a target of a monoclonal antibodyapproved for medical use, such as, for example without limitation, theCD20 epitope recognized by rituximab. In some embodiments, the epitopecomprises the amino acid sequence shown in SEQ ID NO: 397.

(SEQ ID NO: 397) CPYSNPSLCIn some embodiments, the epitope is located within the CAR. For examplewithout limitation, the epitope can be located between the scFv and thehinge of a CAR. In some embodiments, two instances of the same epitope,separate by linkers, may be used in the CAR. For example, thepolypeptide comprising the amino acid sequence shown in SEQ ID NO: 398can be used within a CAR, located between the light chain variableregion and the hinge.

(SEQ ID NO: 398) GSGGGGSCPYSNPSLCSGGGGSCPYSNPSLCSGGGGS

In some embodiments, the epitope-specific antibody may be conjugatedwith a cytotoxic drug. It is also possible to promote CDC cytotoxicityby using engineered antibodies on which are grafted component(s) of thecomplement system. In some embodiments, activation of the CAR-T cellscan be modulated by depleting the cells using an antibody whichrecognizes the epitope.

Therapeutic Applications

Isolated cells obtained by the methods described above, or cell linesderived from such isolated cells, can be used as a medicament. In someembodiments, such a medicament can be used for treating cancer. In someembodiments, the cancer is multiple myeloma malignant plasma cellneoplasm, Hodgkin's lymphoma, nodular lymphocyte predominant Hodgkin'slymphoma, Kahler's disease and Myelomatosis, plasma cell leukemia,plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia,B-cell non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML),chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL),chronic myeloid leukemia (CML), follicular lymphoma, Burkitt's lymphoma,marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma,precursor B-lymphoblastic lymphoma, myeloid leukemia, Waldenstrom'smacroglobulienemia, diffuse large B cell lymphoma, follicular lymphoma,marginal zone lymphoma, mucosa-associated lymphatic tissue lymphoma,small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma,primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmactyiclymphoma, Waldenström macroglobulinemia, nodal marginal zone B celllymphoma, splenic marginal zone lymphoma, intravascular large B-celllymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, Tcell/histiocyte-rich large B-cell lymphoma, primary central nervoussystem lymphoma, primary cutaneous diffuse large B-cell lymphoma (legtype), EBV positive diffuse large B-cell lymphoma of the elderly,diffuse large B-cell lymphoma associated with inflammation,intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma,plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associatedmulticentric Castleman disease, B-cell lymphoma unclassified withfeatures intermediate between diffuse large B-cell lymphoma and Burkittlymphoma, B-cell lymphoma unclassified with features intermediatebetween diffuse large B-cell lymphoma and classical Hodgkin lymphoma, oranother B-cell related lymphomas.

In some embodiments, an isolated cell according to the invention, orcell line derived from the isolated cells, can be used in themanufacture of a medicament for treatment of a cancer in a patient inneed thereof.

Also provided herein are methods for treating patients. In someembodiments the method comprises. providing an immune cell of theinvention to a patient in need thereof. In some embodiments, the methodcomprises a step of administrating transformed immune cells of theinvention to a patient in need thereof.

In some embodiments, T cells of the invention can undergo robust in vivoT cell expansion and can persist for an extended amount of time.

Methods of treatment of the invention can be ameliorating, curative orprophylactic. The method of the invention may be either part of anautologous immunotherapy or part of an allogenic immunotherapytreatment. The invention is particularly suitable for allogeneicimmunotherapy. T cells from donors can be transformed intonon-alloreactive cells using standard protocols and reproduced asneeded, thereby producing CAR-T cells which may be administered to oneor several patients. Such CAR-T cell therapy can be made available as an“off the shelf” therapeutic product.

Cells that can be used with the disclosed methods are described in theprevious section. Treatment can be used to treat patients diagnosedwith, for example, cancer. Cancers that may be treated include, forexample without limitation, cancers that involve B lymphocytes,including any of the above-listed cancers. Types of cancers to betreated with the CARs and CAR-T cells of the invention include, but arenot limited to certain leukemia or lymphoid malignancies. Adulttumors/cancers and pediatric tumors/cancers are also included. In someembodiments, the treatment can be in combination with one or moretherapies against cancer selected from the group of antibodies therapy,chemotherapy, cytokines therapy, dendritic cell therapy, gene therapy,hormone therapy, laser light therapy and radiation therapy.

In some embodiments, treatment can be administrated into patientsundergoing an immunosuppressive treatment. Indeed, the inventionpreferably relies on cells or population of cells, which have been maderesistant to at least one immunosuppressive agent due to theinactivation of a gene encoding a receptor for such immunosuppressiveagent. In this aspect, the immunosuppressive treatment should help theselection and expansion of the T cells according to the invention withinthe patient. The administration of the cells or population of cellsaccording to the invention may be carried out in any convenient manner,including by aerosol inhalation, injection, ingestion, transfusion,implantation or transplantation. The compositions described herein maybe administered to a patient subcutaneously, intradermaliy,intratumorally, intranodally, intramedullary, intramuscularly, byintravenous or intralymphatic injection, or intraperitoneally. In oneembodiment, the cell compositions of the invention are preferablyadministered by intravenous injection.

In some embodiments the administration of the cells or population ofcells can comprise administration of, for example, about 10⁴ to about10⁹ cells per kg body weight including all integer values of cellnumbers within those ranges. In some embodiments the administration ofthe cells or population of cells can comprise administration of about10⁵ to 10⁶ cells per kg body weight including all integer values of cellnumbers within those ranges. The cells or population of cells can beadministrated in one or more doses. In some embodiments, said effectiveamount of cells can be administrated as a single dose. In someembodiments, said effective amount of cells can be administrated as morethan one dose over a period time. Timing of administration is within thejudgment of managing physician and depends on the clinical condition ofthe patient. The cells or population of cells may be obtained from anysource, such as a blood bank or a donor. While individual needs vary,determination of optimal ranges of effective amounts of a given celltype for a particular disease or conditions within the skill of the art.An effective amount means an amount which provides a therapeutic orprophylactic benefit. The dosage administrated will be dependent uponthe age, health and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment and the nature of the effectdesired. In some embodimetns, an effective amount of cells orcomposition comprising those cells are administrated parenterally. Insome embodiments, administration can be an intravenous administration.In some embodimetns, administration can be directly done by injectionwithin a tumor.

In some embodiments of the invention, cells are administered to apatient in conjunction with (e.g., before, simultaneously or following)any number of relevant treatment modalities, including but not limitedto treatment with agents such as monoclonal antibody therapy, CCR2antagonist (e.g., INC-8761), antiviral therapy, cidofovir andinterleukin-2, Cytarabine (also known as ARA-C) or nataliziimabtreatment for MS patients or efaliztimab treatment for psoriasispatients or other treatments for PML patients. In some embodiments, BCMAspecific CAR-T cells are administered to a patient in conjunction withone or more of the following: an anti-PD-1 antibody (e.g., nivolumab,pembrolizumab, or PF-06801591), an anti-PD-L1 antibody (e.g., avelumab,atezolizumab, or durvalumab), an anti-OX40 antibody (e.g., PF-04518600),an anti-4-1BB antibody (e.g., PF-05082566), an anti-MCSF antibody (e.g.,PD-0360324), an anti-GITR antibody, and/or an anti-TIGIT antibody. Insome embodiments, a BCMA specific CAR comprising the amino acid sequenceshown in SEQ ID NO: 396 is administered to a patient in conjunction withanti-PD-L1 antibody avelumab. In further embodiments, the T cells of theinvention may be used in combination with chemotherapy, radiation,immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as CAMPATH, anti-CD3 antibodies or otherantibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin,mycoplienolic acid, steroids, FR901228, cytokines, and/or irradiation.These drugs inhibit either the calcium dependent phosphatase calcineurin(cyclosporine and FK506) or inhibit the p70S6 kinase that is importantfor growth factor induced signaling (rapamycin) (Henderson, Naya et al.1991; Liu, Albers et al. 1992; Bierer, Hollander et al. 1993). In afurther embodiment, the cell compositions of the invention areadministered to a patient in conjunction with (e.g., before,simultaneously or following) bone marrow transplantation, T cellablative therapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH, In some embodiments, the cell compositions ofthe invention are administered following B-cell ablative therapy such asagents that react with CD20, e.g., Rituxan. For example, in oneembodiment, subjects may undergo standard treatment with high dosechemotherapy followed by peripheral blood stem cell transplantation. Incertain embodiments, following the transplant, subjects receive aninfusion of the expanded immune cells of the invention. In someembodiments, expanded cells are administered before or followingsurgery.

Kits

The invention also provides kits for use in the instant methods. Kits ofthe invention include one or more containers comprising a polynucleotideencoding a BCMA specific CAR, or an engineered immune cell comprising apolynucleotide encoding a BCMA specific CAR as described herein, andinstructions for use in accordance with any of the methods of theinvention described herein. Generally, these instructions comprise adescription of administration of the engineered immune cell for theabove described therapeutic treatments.

The instructions relating to the use of the engineered immune cells asdescribed herein generally include information as to dosage, dosingschedule, and route of administration for the intended treatment. Thecontainers may be unit doses, bulk packages (e.g., multi-dose packages)or sub-unit doses. Instructions supplied in the kits of the inventionare typically written instructions on a label or package insert (e.g., apaper sheet included in the kit), but machine-readable instructions(e.g., instructions carried on a magnetic or optical storage disk) arealso acceptable.

The kits of this invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Alsocontemplated are packages for use in combination with a specific device,such as an inhaler, nasal administration device (e.g., an atomizer) oran infusion device such as a minipump. A kit may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Thecontainer may also have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is a BCMA antibody. The container may further comprise asecond pharmaceutically active agent.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the invention in any way. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and fall within the scope of the appendedclaims.

Representative materials of the present invention were deposited in theAmerican Type Culture Collection (ATCC) on February 9, 2016. Thebiological deposit having ATCC Accession No. PTA-122834 is a vectorcomprising a polynucleotide encoding a BCMA specific CAR. The depositwas made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Pfizer, Inc. and ATCC, which assures permanent and unrestrictedavailability of the progeny of the culture of the deposit to the publicupon issuance of the pertinent U.S. patent or upon laying open to thepublic of any U.S. or foreign patent application, whichever comes first,and assures availability of the progeny to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 U.S.C. Section 122 and the Commissioner's rules pursuant thereto(including 37 C.F.R. Section 1.14 with particular reference to 886 OG638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

EXAMPLES Example 1 Determination of Kinetics and Affinity of BCMA/HumanIgG Interactions at 25° C. and/or 37° C.

This example determines the kinetics and affinity of various anti-BCMAantibodies at 25° C. and 37° C.

All experiments were performed on a Bio-Rad Proteon XPR36 surfacePlasmon resonance biosensor (Bio-Rad, Hercules, Calif.). An array ofanti-BCMA antibodies was prepared using an amine-coupling method on aBio-Rad GLC Sensor Chip similar to that described in Abdiche, et al.,Anal. Biochem. 411, 139-151 (2011). The analysis temperature for theimmobilization was 25° C. and the running buffer was HBS-T+(10 mM HEPES,150 mM NaCl, 0.05% Tween-20, pH 7.4). Channels were activated in theanalyte (horizontal) direction by injecting a mixture of 1 mM ECD and0.25 mM NHS for 3 minutes at a flow rate of 30 μL/min. IgGs wereimmobilized on the activated spots by injecting them in the ligand(vertical) direction at 20 μg/mL in 10 mM Acetate pH 4.5 buffer for 1.5minutes at 30 μg/m L. The activated surfaces were blocked by injecting1M ethanolamine, pH 8.5 in the analyte direction for 3 minutes at 30μL/min.

The analysis temperature for the BCMA binding analysis was 37° C. or 25°C. in a running buffer of HBS-T+, supplemented with 1 mg/mL BSA. Akinetic titration method was employed for the interaction analysis asdescribed in Abdiche, et al. Human BCMA (huBCMA) or cynomolgus monkeyBCMA (cyBCMA) analyte was injected in the analyte direction using aseries of injections from low to high concentration. The concentrationsused were 0.08 nM, 0.4 nM, 2 nM, 10 nM and 50 nM (a 5-membered series,with a 5-fold dilution factor and top concentration of 50 nM). Theassociation time for a given analyte dilution was two minutes.Immediately after the 50 nM BCMA injection, dissociation was monitoredfor 2 hours. Prior to the BCMA analyte injections, buffer was injected 5times using the same association and dissociation times at the BCMAanalyte cycles to prepare a buffer blank sensorgram fordouble-referencing purposes (double referencing as described in Myszka,J. Mol. Recognit. 12, 279-284 (1999).

The sensorgrams were double-referenced and fit to a 1:1 Langmuir withmass transport kinetic titration model in BIAevaluation Software version4.1.1 (GE Lifesciences, Piscataway, N.J.). The kinetics and affinityparameters for various anti-BCMA antibodies of the invention are shownin Tables 4A-4C. The antibodies shown in Tables 4A-4C share the same VHand VL regions as the CARs shown in Table 1 having the same name.

TABLE 4A K_(D) Sample k_(a) (1/Ms) k_(d) (1/s) t_(1/2) (min) (pM)A02_Rd4_6nM_C01 1.2E+06 2.8E−05 411 24 A02_Rd4_6nM_C16 1.1E+06 6.2E−05187 59 Combo_Rd4_0.6nM_C29 6.6E+06 1.4E−04 83 21 L3PY/H3TAQ 2.6E+061.4E−04 84 53

TABLE 4B ka (1/Ms) huBCMA kd (1/s) huBCMA T½ (min) to KD (nM) toAntibody @ 25° C. @ 25° C. huBCMA @ 25° C. huBCMA @ 25° C. P6E01/P6E011.04E+06 4.15E−03 2.8 4.0 P6E01/H3.AQ 8.35E+05 3.45E−04 33.53 0.41L1.LGF/L3.KW/P6E01 8.31E+05 7.55E−03 1.53 9.08 L1.LGF/L3.NY/P6E011.33E+06 4.40E−03 2.63 3.32 L1.GDF/L3.NY/P6E01 1.60E+06 5.92E−03 1.953.70 L1.LGF/L3.KW/H3.AL 4.28E+05 1.23E−03 9.40 2.87 L1.LGF/L3.KW/H3.AP9.28E+05 2.27E−03 5.10 2.44 L1.LGF/L3.KW/H3.AQ 5.24E+05 9.56E−04 12.091.82 L1.LGF/L3.PY/H3.AP 4.57E+05 9.69E−04 11.92 2.12 L1.LGF/L3.PY/H3.AQ9.31E+05 8.86E−04 13.04 0.95 L1.LGF/L3.NY/H3.AL 7.63E+05 9.70E−04 11.911.27 L1.LGF/L3.NY/H3.AP 9.36E+05 5.33E−04 21.67 0.57 L1.LGF/L3.NY/H3.AQ6.66E+05 2.99E−04 38.61 0.45 L1.GDF/L3.KW/H3.AL 4.45E+05 3.90E−03 2.968.76 L1.GDF/L3.KW/H3.AP 1.17E+06 4.61E−03 2.51 3.93 L1.GDF/L3.KW/H3.AQ7.97E+05 3.48E−03 3.32 4.37 L1.GDF/L3.PY/H3.AQ 1.42E+06 1.35E−02 0.869.49 L1.GDF/L3.NY/H3.AL 9.07E+05 4.03E−03 2.87 4.44 L1.GDF/L3.NY/H3.AP1.41E+06 1.41E−03 8.21 1.00 L1.GDF/L3.NY/H3.AQ 9.84E+05 7.22E−04 16.000.73 L3.KW/P6E01 7.40E+05 3.15E−04 36.66 0.43 L3.PY/P6E01 7.12E+052.28E−04 50.74 0.32 L3.NY/P6E01 8.76E+05 3.84E−04 30.08 0.44 ka (1/Ms)huBCMA kd (1/s) huBCMA T½ (min) to KD (nM) to Antibody @ 37° C. @ 37° C.huBCMA @ 37° C. huBCMA @ 37° C. L3.PY/L1.PS/P6E01 2.49E+06 1.13E−0310.21 0.45 L3.PY/L1.AH/P6E01 2.55E+06 1.26E−03 9.19 0.49L3.PY/L1.FF/P6E01 2.39E+06 1.41E−03 8.18 0.59 L3.PY/L1.PH/P6E01 2.81E+069.13E−04 12.65 0.32 L3.PY/L3.KY/P6E01 3.18E+06 1.09E−03 10.65 0.34L3.PY/L3.KF/P6E01 2.88E+06 2.08E−03 5.56 0.72 L3.PY/H2.QR 2.56E+061.19E−03 9.75 0.46 L3.PY/H2.DY 2.60E+06 1.38E−03 8.37 0.53 L3.PY/H2.YQ2.58E+06 1.56E−03 7.41 0.60 L3.PY/H2.LT 2.40E+06 1.29E−03 8.95 0.54L3.PY/H2.HA 2.43E+06 1.47E−03 7.89 0.60 L3.PY/H2.QL 2.64E+06 2.18E−035.31 0.82 L3.PY/H3.YA 3.15E+06 1.18E−03 9.82 0.37 L3.PY/H3.AE 3.29E+061.39E−03 8.32 0.42 L3.PY/H3.AQ 3.08E+06 1.73E−03 6.69 0.56 L3.PY/H3.TAQ3.08E+06 1.14E−03 10.13 0.37 L3.PY/P6E01 2.65E+06 1.96E−03 5.91 0.74L3.PY/L1.PS/H2.QR 3.97E+06 1.03E−01 0.11 25.85 L3.PY/L1.PS/H2.DY3.22E+06 3.61E−03 3.20 1.12 L3.PY/L1.PS/H2.YQ 3.35E+06 4.30E−03 2.691.28 L3.PY/L1.PS/H2.LT 3.40E+06 4.65E−03 2.49 1.37 L3.PY/L1.PS/H2.HA3.30E+06 1.06E−02 1.09 3.21 L3.PY/L1.PS/H2.QL 1.52E+07 3.14E−01 0.0420.64 L3.PY/L1.PS/H3.YA 3.07E+06 9.05E−03 1.28 2.95 L3.PY/L1.PS/H3.AE3.14E+06 1.46E−03 7.93 0.46 L3.PY/L1.PS/H3.AQ 3.26E+06 1.79E−03 6.460.55 L3.PY/L1.PS/H3.TAQ 3.25E+06 2.46E−03 4.70 0.76 L3.PY/L1.AH/H2.QR3.13E+06 1.81E−03 6.39 0.58 L3.PY/L1.AH/H2.DY 3.05E+06 1.52E−03 7.620.50 L3.PY/L1.AH/H2.YQ 2.42E+06 1.93E−03 6.00 0.80 L3.PY/L1.AH/H2.LT3.16E+06 1.23E−03 9.38 0.39 L3.PY/L1.AH/H2.HA 3.33E+06 1.81E−03 6.370.54 L3.PY/L1.AH/H2.QL 3.04E+06 1.60E−03 7.22 0.53 L3.PY/L1.AH/H3.YA3.00E+06 1.50E−03 7.73 0.50 L3.PY/L1.AH/H3.AE 3.32E+06 1.73E−03 6.700.52 L3.PY/L1.AH/H3.AQ 3.03E+06 1.97E−03 5.85 0.65 L3.PY/L1.AH/H3.TAQ3.27E+06 1.19E−03 9.68 0.37 L3.PY/L1.FF/H2.QR 3.47E+06 1.77E−03 6.540.51 L3.PY/L1.FF/H2.DY 4.14E+06 2.71E−03 4.27 0.65 L3.PY/L1.FF/H2.YQ3.32E+06 1.52E−03 7.61 0.46 L3.PY/L1.FF/H2.LT 3.30E+06 1.67E−03 6.920.51 L3.PY/L1.FF/H2.HA 3.49E+06 2.19E−03 5.29 0.63 L3.PY/L1.FF/H2.QL3.48E+06 1.40E−03 8.28 0.40 L3.PY/L1.FF/H3.YA 3.50E+06 1.80E−03 6.410.51 L3.PY/L1.FF/H3.AE 3.82E+06 2.63E−03 4.39 0.69 L3.PY/L1.FF/H3.AQ3.32E+06 1.54E−03 7.51 0.46 L3.PY/L1.FF/H3.TAQ 3.52E+06 1.89E−03 6.120.54 L3.PY/L1.PH/H2.QR 3.69E+06 2.36E−03 4.89 0.64 L3.PY/L1.PH/H2.HA2.37E+06 1.16E−03 9.99 0.49 L3.PY/L1.PH/H3.AE 3.68E+06 1.34E−03 8.610.36 L3.PY/L1.PH/H3.AQ 3.08E+06 1.59E−03 7.27 0.52 L3.PY/L1.PH/H3.TAQ3.58E+06 2.13E−03 5.43 0.59 L3.PY/L3.KY/H2.QR 2.95E+06 9.90E−04 11.670.34 L3.PY/L3.KY/H2.DY 3.19E+06 6.42E−04 18.00 0.20 L3.PY/L3.KY/H2.YQ2.14E+06 1.65E−03 7.02 0.77 L3.PY/L3.KY/H2.LT 2.92E+06 9.06E−04 12.750.31 L3.PY/L3.KY/H2.HA 3.29E+06 1.63E−03 7.10 0.49 L3.PY/L3.KY/H2.QL3.65E+06 2.08E−03 5.56 0.57 L3.PY/L3.KY/H3.YA 3.30E+06 9.12E−04 12.670.28 L3.PY/L3.KY/H3.TAQ 2.79E+06 6.49E−04 17.79 0.23 L3.PY/L3.KF/H2.DY2.74E+06 1.82E−03 6.35 0.67 L3.PY/L3.KF/H2.YQ 1.96E+06 2.23E−03 5.181.14 L3.PY/L3.KF/H2.LT 2.75E+06 1.91E−03 6.05 0.69 L3.PY/L3.KF/H2.QL2.07E+06 1.25E−03 9.26 0.60 L3.PY/L3.KF/H3.YA 3.12E+06 1.47E−03 7.850.47 L3.PY/L3.KF/H3.AE 3.07E+06 1.55E−03 7.44 0.51 L3.PY/L3.KF/H3.AQ3.48E+06 2.27E−03 5.09 0.65 L3.PY/L3.KF/H3.TAQ 2.82E+06 1.62E−03 7.120.58 ka (1/Ms) cyBCMA kd (1/s) cyBCMA T½ (min) to KD (nM) to Antibody @25° C. @ 25° C. cyBCMA @ 25° C. cyBCMA @ 25° C. P6E01/P6E01 7.02E−020.16 115.4 P6E01/H3.AQ 1.08E+06 7.40E−03 1.6 6.9 L1.LGF/L3.KW/P6E014.55E+05 1.95E−02 0.6 42.8 L1.LGF/L3.NY/P6E01 9.20E+05 1.05E−02 1.1 11.4L1.GDF/L3.NY/P6E01 1.20E+06 7.67E−03 1.5 6.4 L1.LGF/L3.KW/H3.AL 2.90E+051.21E−02 1.0 41.8 L1.LGF/L3.KW/H3.AP 5.54E+05 1.54E−02 0.7 27.8L1.LGF/L3.KW/H3.AQ 5.27E+05 3.55E−03 3.3 6.7 L1.LGF/L3.PY/H3.AP 3.64E+051.30E−02 0.9 35.8 L1.LGF/L3.PY/H3.AQ 1.00E+06 4.77E−03 2.4 4.8L1.LGF/L3.NY/H3.AL 6.35E+05 1.48E−02 0.8 23.2 L1.LGF/L3.NY/H3.AP8.30E+05 5.57E−03 2.1 6.7 L1.LGF/L3.NY/H3.AQ 7.51E+05 1.48E−03 7.8 2.0L1.GDF/L3.KW/H3.AL 3.18E+05 1.80E−02 0.6 56.7 L1.GDF/L3.KW/H3.AP8.14E+05 2.03E−02 0.6 24.9 L1.GDF/L3.KW/H3.AQ 8.02E+05 5.65E−03 2.0 7.0L1.GDF/L3.PY/H3.AQ 1.55E+06 1.66E−02 0.7 10.7 L1.GDF/L3.NY/H3.AL9.00E+05 2.19E−02 0.5 24.3 L1.GDF/L3.NY/H3.AP 1.36E+06 7.02E−03 1.6 5.2L1.GDF/L3.NY/H3.AQ 1.18E+06 1.36E−03 8.5 1.2 L3.KW/P6E01 7.63E+052.57E−03 4.5 3.4 L3.PY/P6E01 8.55E+05 2.93E−03 3.9 3.4 L3.NY/P6E011.01E+06 2.87E−03 4.0 2.8 ka (1/Ms) cyBCMA kd (1/s) cyBCMA T½ (min) toKD (nM) to Antibody @ 37° C. @ 37° C. cyBCMA @ 37° C. cyBCMA @ 37° C.L3.PY/L1.PS/P6E01 2.17E+06 6.06E−03 1.91 2.79 L3.PY/L1.AH/P6E01 2.16E+065.72E−03 2.02 2.65 L3.PY/L1.FF/P6E01 2.45E+06 5.91E−03 1.96 2.41L3.PY/L1.PH/P6E01 2.17E+06 7.89E−03 1.46 3.63 L3.PY/L3.KY/P6E01 2.27E+065.02E−03 2.30 2.21 L3.PY/L3.KF/P6E01 2.39E+06 8.30E−03 1.39 3.48L3.PY/H2.QR 2.18E+06 6.58E−03 1.76 3.02 L3.PY/H2.DY 2.24E+06 6.18E−031.87 2.76 L3.PY/H2.YQ 2.46E+06 6.21E−03 1.86 2.53 L3.PY/H2.LT 2.09E+067.57E−03 1.53 3.63 L3.PY/H2.HA 1.99E+06 7.55E−03 1.53 3.79 L3.PY/H2.QL2.05E+06 1.26E−02 0.91 6.16 L3.PY/H3.YA 2.87E+06 5.40E−03 2.14 1.88L3.PY/H3.AE 2.82E+06 5.04E−03 2.29 1.79 L3.PY/H3.AQ 2.77E+06 5.39E−032.14 1.94 L3.PY/H3.TAQ 2.57E+06 4.37E−03 2.64 1.70 L3.PY/P6E01 2.20E+061.31E−02 0.88 5.96 L3.PY/L1.PS/H2.QR 5.25E+05 6.70E−04 17.23 1.28L3.PY/L1.PS/H2.DY 1.90E+06 3.78E−03 3.06 1.99 L3.PY/L1.PS/H2.YQ 2.00E+063.74E−03 3.09 1.87 L3.PY/L1.PS/H2.LT 2.17E+06 4.11E−03 2.81 1.89L3.PY/L1.PS/H2.HA 1.45E+06 2.69E−03 4.30 1.86 L3.PY/L1.PS/H2.QL 6.57E+056.36E−04 18.17 0.97 L3.PY/L1.PS/H3.YA 1.77E+06 9.98E−03 1.16 5.65L3.PY/L1.PS/H3.AE 2.46E+06 4.13E−03 2.80 1.68 L3.PY/L1.PS/H3.AQ 2.52E+064.33E−03 2.67 1.72 L3.PY/L1.PS/H3.TAQ 2.58E+06 5.52E−03 2.09 2.14L3.PY/L1.AH/H2.QR 2.20E+06 4.91E−03 2.35 2.23 L3.PY/L1.AH/H2.DY 2.32E+064.51E−03 2.56 1.95 L3.PY/L1.AH/H2.YQ 1.58E+06 4.31E−03 2.68 2.74L3.PY/L1.AH/H2.LT 2.19E+06 2.96E−03 3.91 1.35 L3.PY/L1.AH/H2.HA 2.58E+064.39E−03 2.63 1.70 L3.PY/L1.AH/H2.QL 2.62E+06 9.55E−03 1.21 3.65L3.PY/L1.AH/H3.YA 2.37E+06 5.26E−03 2.20 2.22 L3.PY/L1.AH/H3.AE 2.25E+063.56E−03 3.25 1.58 L3.PY/L1.AH/H3.AQ 2.24E+06 3.99E−03 2.90 1.78L3.PY/L1.AH/H3.TAQ 2.28E+06 3.02E−03 3.83 1.32 L3.PY/L1.FF/H2.QR2.55E+06 4.21E−03 2.75 1.65 L3.PY/L1.FF/H2.DY 2.66E+06 5.00E−03 2.311.88 L3.PY/L1.FF/H2.YQ 2.19E+06 3.26E−03 3.55 1.49 L3.PY/L1.FF/H2.LT2.19E+06 3.41E−03 3.38 1.56 L3.PY/L1.FF/H2.HA 2.33E+06 4.17E−03 2.771.79 L3.PY/L1.FF/H2.QL 2.36E+06 4.49E−03 2.57 1.91 L3.PY/L1.FF/H3.YA2.46E+06 4.16E−03 2.77 1.69 L3.PY/L1.FF/H3.AE 2.85E+06 5.01E−03 2.311.76 L3.PY/L1.FF/H3.AQ 2.18E+06 3.29E−03 3.51 1.51 L3.PY/L1.FF/H3.TAQ2.32E+06 3.76E−03 3.07 1.62 L3.PY/L1.PH/H2.QR 2.42E+06 4.36E−03 2.651.80 L3.PY/L1.PH/H2.HA 1.61E+06 5.53E−03 2.09 3.44 L3.PY/L1.PH/H3.AE2.61E+06 2.02E−03 5.72 0.77 L3.PY/L1.PH/H3.AQ 2.28E+06 3.41E−03 3.391.50 L3.PY/L1.PH/H3.TAQ 2.51E+06 3.20E−03 3.61 1.28 L3.PY/L3.KY/H2.QR2.05E+06 7.74E−03 1.49 3.78 L3.PY/L3.KY/H2.DY 1.96E+06 2.43E−03 4.751.24 L3.PY/L3.KY/H2.YQ 1.27E+06 2.58E−03 4.47 2.04 L3.PY/L3.KY/H2.LT1.82E+06 2.32E−03 4.98 1.27 L3.PY/L3.KY/H2.HA 2.28E+06 3.18E−03 3.631.40 L3.PY/L3.KY/H2.QL 2.75E+06 4.09E−03 2.83 1.49 L3.PY/L3.KY/H3.YA1.84E+06 4.28E−03 2.70 2.33 L3.PY/L3.KY/H3.TAQ 1.81E+06 1.92E−03 6.031.06 L3.PY/L3.KF/H2.DY 2.08E+06 3.68E−03 3.14 1.77 L3.PY/L3.KF/H2.YQ1.41E+06 5.01E−03 2.30 3.55 L3.PY/L3.KF/H2.LT 1.91E+06 4.13E−03 2.802.16 L3.PY/L3.KF/H2.QL 1.42E+06 3.10E−03 3.73 2.18 L3.PY/L3.KF/H3.YA2.10E+06 7.96E−03 1.45 3.78 L3.PY/L3.KF/H3.AE 1.85E+06 5.64E−03 2.053.05 L3.PY/L3.KF/H3.AQ 2.55E+06 2.38E−03 4.85 0.93 L3.PY/L3.KF/H3.TAQ2.01E+06 1.91E−03 6.05 0.95

TABLE 4C Human BCMA Cyno BCMA ka kd KD ka kd KD Antibody (1/Ms) (1/s)(pM) (1/Ms) (1/s) (pM) P5A2_VHVL (P5A) 6.96E+06 3.87E−02 5567 1.61E+061.64E−02 10230 A02_Rd4_0.6nM_C06 3.49E+06 7.37E−05 21 1.81E+06 1.05E−0458 A02_Rd4_0.6nM_C09 5.50E+06 9.75E−05 18 2.13E+06 1.74E−04 82A02_Rd4_6nM_C16 (P5AC16) 1.56E+06 1.41E−04 90 1.34E+06 1.58E−04 118A02_Rd4_6nM_C03 1.69E+06 1.26E−04 75 1.17E+06 1.85E−04 158A02_Rd4_6nM_C01 3.11E+06 9.20E−05 30 1.45E+06 5.83E−04 401A02_Rd4_6nM_C26 4.26E+06 1.39E−04 33 2.21E+06 4.48E−04 203A02_Rd4_6nM_C25 2.75E+06 1.80E−04 65 1.50E+06 3.30E−04 220A02_Rd4_6nM_C22 3.38E+06 1.82E−04 54 1.84E+06 3.24E−04 176A02_Rd4_6nM_C19 3.00E+06 1.48E−04 49 2.54E+06 6.61E−04 260A02_Rd4_0.6nM_C03 4.27E+06 1.82E−04 43 2.12E+06 4.26E−04 201A02_Rd4_6nM_C07 1.48E+06 1.89E−04 128 6.91E+05 7.86E−04 1138A02_Rd4_6nM_C23 1.22E+07 2.55E−04 21 2.63E+06 4.14E−04 157A02_Rd4_0.6nM_C18 4.73E+06 2.29E−04 48 3.24E+06 6.39E−04 197A02_Rd4_6nM_C10 4.51E+06 3.15E−04 70 1.90E+06 8.98E−04 472A02_Rd4_6nM_C05 3.10E+06 3.08E−04 99 1.36E+06 1.29E−03 950A02_Rd4_0.6nM_C10 2.30E+06 2.96E−04 129 8.83E+05 1.63E−03 1842A02_Rd4_6nM_C04 4.47E+06 6.03E−04 135 2.18E+06 8.31E−04 381A02_Rd4_0.6nM_C26 7.26E+06 4.43E−04 61 2.71E+06 2.56E−03 941A02_Rd4_0.6nM_C13 8.53E+06 5.66E−04 66 2.29E+06 1.28E−03 560A02_Rd4_0.6nM_C01 (P5AC1) 4.74E+06 9.15E−04 193 2.39E+06 1.57E−03 655A02_Rd4_6nM_C08 3.92E+06 7.38E−04 188 2.23E+06 1.13E−02 5072 P5C1_VHVL(PC1) 1.16E+07 6.92E−02 5986 3.53E+06 5.38E−02 15231 C01_Rd4_6nM_C247.47E+06 3.48E−03 467 3.17E+06 8.91E−04 281 C01_Rd4_6nM_C26 1.50E+071.36E−03 90 4.75E+06 1.99E−03 419 C01_Rd4_6nM_C02 1.61E+07 1.44E−03 895.12E+06 2.18E−03 426 C01_Rd4_6nM_C10 1.31E+07 2.12E−03 162 4.44E+062.19E−03 493 C01_Rd4_0.6nM_C27 1.23E+07 3.74E−03 303 3.34E+06 2.85E−03852 C01_Rd4_6nM_C20 6.02E+06 2.76E−03 459 3.60E+06 6.25E−03 1737C01_Rd4_6nM_C12 1.21E+07 6.49E−03 535 4.51E+06 3.70E−03 820C01_Rd4_0.6nM_C16 1.55E+07 6.30E−03 407 4.95E+06 4.64E−03 939C01_Rd4_0.6nM_C09 1.51E+07 8.25E−03 545 5.28E+06 9.36E−03 1773C01_Rd4_6nM_C09 1.58E+07 1.28E−02 811 3.73E+06 8.68E−03 2328C01_Rd4_0.6nM_C03 1.55E+07 1.50E−02 964 4.72E+06 1.19E−02 2528C01_Rd4_0.6nM_C06 1.82E+07 1.54E−02 847 6.22E+06 1.21E−02 1948C01_Rd4_6nM_C04 2.33E+07 4.97E−02 2134 6.34E+06 3.27E−02 5156COMBO_Rd4_0.6nM_C22 1.97E+06 7.15E−05 36 1.34E+06 6.66E−05 50COMBO_Rd4_6nM_C21 1.17E+07 7.34E−05 6 3.17E+06 2.48E−04 78COMBO_Rd4_6nM_C10 5.47E+06 9.72E−05 18 1.52E+06 1.60E−04 105COMBO_Rd4_0.6nM_C04 1.07E+07 1.58E−04 15 3.52E+06 1.37E−04 39COMBO_Rd4_6nM_C25 7.98E+06 1.13E−04 14 2.85E+06 2.26E−04 79COMBO_Rd4_0.6nM_C21 1.34E+07 1.15E−04 9 3.63E+06 3.04E−04 84COMBO_Rd4_6nM_C11 6.74E+06 1.24E−04 18 2.64E+06 4.12E−04 156COMBO_Rd4_0.6nM_C20 7.65E+06 1.46E−04 19 3.09E+06 2.84E−04 92COMBO_Rd4_6nM_C09 8.85E+06 1.43E−04 16 2.37E+06 3.18E−04 134COMBO_Rd4_6nM_C08 8.99E+06 1.69E−04 19 3.06E+06 4.28E−04 140COMBO_Rd4_0.6nM_C19 7.86E+06 1.55E−04 20 2.92E+06 9.79E−04 336COMBO_Rd4_0.6nM_C02 8.57E+06 1.85E−04 22 3.01E+06 4.94E−04 164COMBO_Rd4_0.6nM_C23 7.39E+06 2.10E−04 28 2.81E+06 5.31E−04 189COMBO_Rd4_0.6nM_C29 1.47E+07 2.77E−04 19 4.00E+06 3.36E−04 84COMBO_Rd4_0.6nM_C09 1.04E+07 3.19E−04 31 3.77E+06 3.46E−04 92COMBO_Rd4_6nM_C12 (PC1C12) 1.38E+07 2.70E−04 20 3.29E+06 4.86E−04 148COMBO_Rd4_0.6nM_C30 4.35E+06 2.82E−04 65 1.68E+06 8.08E−04 481COMBO_Rd4_0.6nM_C14 8.66E+06 3.28E−04 38 3.48E+06 6.45E−04 185COMBO_Rd4_6nM_C07 1.05E+07 3.71E−04 35 3.94E+06 9.34E−04 237COMBO_Rd4_6nM_C02 1.05E+06 4.43E−04 422 7.95E+05 1.36E−03 1714COMBO_Rd4_0.6nM_C05 4.32E+06 4.97E−04 115 1.94E+06 1.72E−03 886COMBO_Rd4_0.6nM_C17 8.68E+06 8.01E−04 92 3.06E+06 1.01E−03 330COMBO_Rd4_6nM_C22 (COM22) 3.03E+06 7.75E−04 256 1.70E+06 1.65E−03 972COMBO_Rd4_0.6nM_C11 5.11E+06 1.06E−03 207 2.20E+06 4.23E−03 1924

Example 2 BCMA Specific CAR-T Cells

This example demonstrates functional activity of BCMA specific CAR-Tcells against BCMA positive (BCMA+) tumor cells.

Among all the BCMA specific CAR molecules generated, eight were selectedfor further activity tests based on affinity to BCMA, cross-reactivityto human BCMA and cyno BCMA, and epitope. The CAR molecules testedincluded: P5A, P5AC1, P5AC16, PC1, PC1C12, COM22, P6DY, and P6AP. Threedifferent architectures were designed: version 1 (v1) comprises anFcγRIIIα hinge, version 2 (v2) comprises a CD8α hinge, and version 3(v3) comprises and IgG1 hinge. The chimeric antigen receptors (CARs)shown in Table 5 were prepared and used and assessed for theirdegranulation activity towards BCMA+ cells. Degranulation activity wasdetermined upon transient expression of each CAR in human T cells.

TABLE 5 Exemplary BCMA specific CARs CAR CAR Amino Acid SequenceComponents P5A-V1 MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCACD8α signal peptide; ASGFTFSSYAMNWVRQAPGKGLEWVSAISDSGGSTYYADSVKP5A2_VHVL VH (Table GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQ 1 SEQ IDNO: 33); GTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATL GS linker;SCRASQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFS P5A2_VHVL VL (SEQ IDGSGSGTDFTLTISRLEPEDFAVYYCQQYGSWPLTFGQGTKVEIK NO: 34);GLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL FcγRIIIα hinge;LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAD CD8α TM domain;APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR 41BB ISD;RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ CD3ζ ζ ISDGLSTATKDTYDALHMQALPPR (SEQ ID NO: 343) P5A-V2MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA CD8α signal peptide;ASGFTFSSYAMNWVRQAPGKGLEWVSAISDSGGSTYYADSVK P5A2_VHVL VH;GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQ GS linker;GTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATL P5A2_VHVL VL;SCRASQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFS CD8α hinge;GSGSGTDFTLTISRLEPEDFAVYYCQQYGSWPLTFGQGTKVEIK CD8α TM domain;TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC 41BB ISD;DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT CD3ζ ζ ISDTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR(SEQ ID NO: 344)P5A-V3 MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA CD8α signal peptide;ASGFTFSSYAMNWVRQAPGKGLEWVSAISDSGGSTYYADSVK P5A2_VHVL VH;GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQ GS linker;GTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATL P5A2_VHVL VL;SCRASQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFS IgG1 hinge;GSGSGTDFTLTISRLEPEDFAVYYCQQYGSwpriFGQGTKVEIK CD8α TM domain;EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVT 41BB ISD;CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV CD3ζ ISDVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR(SEQ IDNO: 345) P5AC1- MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA CD8α signalpeptide; V1 ASGFTFSSYAMNWVRQAPGKGLEWVSAILsSGGSTYYADSVK A02_Rd4_0.6nM_C01GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQ VH (SEQ ID NO: 72);GTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATL GS linker;SCRGGQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFS A02_Rd4_0.6nM_C01GSGSGTDFTLTISRLEPEDFAVYYCQQYQSWPLTFGQGTKVEIK VL (SEQ ID NO: 73);GLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL FcγRIIIα hinge;LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRvkFSRSAD CD8α TM domain;APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR 41BB ISD;RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ CD3ζ ISD GLSTATKDTYDALHMQALPPR(SEQ ID NO: 346) P5AC1- MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCACD8α signal peptide; V2 ASGFTFSSYAMNWVRQAPGKGLEWVSAILsSGGSTYYADSVKA02_Rd4_0.6nM_C01 GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQ VH;GTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATL GS linker;SCRGGQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFS A02_Rd4_0.6nM_C01GSGSGTDFTLTISRLEPEDFAVYYCQQYQSWPLTFGQGTKVEIK VL;TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC CD8α hinge;DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT CD8α TM domain;TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLY 41BB ISD;NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ CD3ζ ISDKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR (SEQ ID NO: 347)P5AC1- MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA CD8α signal peptide;V2.1 ASGFTFSSYAMNWVRQAPGKGLEWVSAILSSGGSTYYADSVK A02_Rd4_0.6nM_C01GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQ VH;GTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATL GS linker;SCRGGQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFS A02_Rd4_0.6nM_C01GSGSGTDFTLTISRLEPEDFAVYYCQQYQSWPLTFGQGTKVEIK VL; rituximab epitope;GSGGGGSCPYSNPSLCSGGGGSCPYSNPSLCSGGGGSTTTPAP CD8α hinge;RPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA CD8α TM domain;PLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEED 41BB ISD;GCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLG CD3ζ ISDRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR (SEQ ID NO: 396) P5AC1-MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA CD8α signal peptide; V3ASGFTFSSYAMNWVRQAPGKGLEWVSAILsSGGSTYYADSVK A02_Rd4_0.6nM_C01GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQ VH;GTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATL GS linker;SCRGGQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFS A02_Rd4_0.6nM_C01GSGSGTDFTLTISRLEPEDFAVYYCQQYQSWPLTFGQGTKVEIK VL;EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVT IgG1 hinge;CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV CD8α TM domain;VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP 41BB ISD;QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN CD3ζ ISDNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR (SEQ IDNO: 348) P5AC16- MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCACD8α signal peptide; V1 ASGFTFSSYAMNWVRQAPGKGLEWVSAISdFGGSTYYADSVKA02_Rd4_6nM_C16 VH GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQ (SEQ IDNO: 39); GTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATL GS linker;SCRASQSVSDIYLAWYQQKPGQAPRLLMYDASIRATGIPDRFS A02_Rd4_6nM_C16 VLGSGSGTDFTLTISRLEPEDFAVYYCQQYQTWPLTFGQGTKVEIK (SEQ ID NO: 40);GLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL FcγRIIIα hinge;LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVkFSRSAD CD8α TM domain;APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR 41BB ISD;RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ CD3ζ ISD;GLSTATKDTYDALHMQALPPR (SEQ ID NO: 349) P5AC16-MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA CD8α signal peptide; V2ASGFTFSSYAMNWVRQAPGKGLEWVSAISdFGGSTYYADSVK A02_Rd4_6nM_C16GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQ VH;GTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATL GS linker;SCRASQSVSDIYLAWYQQKPGQAPRLLMYDASIRATGIPDRFS A02_Rd4_6nM_C16 VL;GSGSGTDFTLTISRLEPEDFAVYYCQQYQTWPLTFGQGTKVEIK CD8α hinge;TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGANHTRGLDFAC CD8α TM domain;DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT 41BB ISD;TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLY CD3ζ ISDNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR (SEQ ID NO: 350)P5AC16- MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA CD8α signalpeptide; V3 ASGFTFSSYAMNWVRQAPGKGLEWVSAISdFGGSTYYADSVK A02_Rd4_6nM_C16GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQ VH;GTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATL GS linker;SCRASQSVSDIYLAWYQQKPGQAPRLLMYDASIRATGIPDRFS A02_Rd4_6nM_C16 VL;GSGSGTDFTLTISRLEPEDFAVYYCQQYQTWPLTFGQGTKVEIK IgG1 hinge;EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVT CD8α TM domain;CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV 41BB ISD;VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP CD3ζ ISDQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR (SEQ IDNO: 351) PC1-V1 MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA CD8α signalpeptide; ASGFTFSSYPMSWVRQAPGKGLEWVSAIGGSGGSTYYADSVK P5C1_VHVL VH (SEQ IDGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDSWG NO: 76);QGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERA GS linker;TLSCRASQSVSSTYLAWYQQKPGQAPRLLIYDASSRAPGIPDRF P5C1_VHVL VL (SEQ IDSGSGSGTDFTLTISRLEPEDFAVYYCQQYSTSPLTFGQGTKVEIK NO: 77);GLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL FcγRIIIα hinge;LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRvKFSRSAD CD8α TM domain;APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR 41BB ISD;RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ CD3ζ ISD GLSTATKDTYDALHMQALPPR(SEQ ID NO: 352) PC1-V2 MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCACD8α signal peptide; ASGFTFSSYPMSWVRQAPGKGLEWVSAIGGSGGSTYYADSVKP5C1_VHVL VH; GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDSWG GS linker;QGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERA P5C1_VHVL VL;TLSCRASQSVSSTYLAWYQQKPGQAPRLLIYDASSRAPGIPDRF CD8α hinge;SGSGSGTDFTLTISRLEPEDFAVYYCQQYSTSPLTFGQGTKVEIK CD8α TM domain;TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC 41BB ISD;DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT CD3ζ ISDTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR (SEQ ID NQ: 353)PC1-V3 MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA CD8α signal peptide;ASGFTFSSYPMSWVRQAPGKGLEWVSAIGGSGGSTYYADSVK P5C1_VHVL VH;GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDSWG GS linker;QGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERA P5C1_VHVL VL;TLSCRASQSVSSTYLAWYQQKPGQAPRLLIYDASSRAPGIPDRF IgG1 hinge;SGSGSGTDFTLTISRLEPEDFAVYYCQQYSTSPLTFGQGTKVEIK CD8α TM domain;EPKSPDKTHTCPPCPAPPVAGPSVFLFRRKRKDTLNAIARTPEVT 41BB ISD;CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV CD3ζ ISDVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR (SEQ IDNO: 354) PC1C12- MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCACD8α signal peptide; V1 ASGFTFSSYPMSWVRQAPGKGLEWVSAIGgSGGWSYYADSVKC01_Rd4_6nM_C12 VH GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDSWG (SEQ ID NO:83); QGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERA GS linker;TLSCWLSQSVSSTYLAWYQQKPGQAPRLLIYDASSRAPGIPDRF C01_Rd4_6nM_C12 VLSGSGSGTDFTLTISRLEPEDFAVYYCQQYSEWPLTFGQGTKVEIK (SEQ ID NO: 84);GLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKL FcγRIIIα hinge;LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAD CD8α TM domain;APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR 41BB ISD;RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ CD3ζ ISD GLSTATKDTYDALHMQALPPR(SEQ ID NO: 355) PC1C12- MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCACD8α signal peptide; V2 ASGFTFSSYPMSWVRQAPGKGLEWVSAIGgSGGWSYYADSVKC01_Rd4_6nM_C12 GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDSWG VH;QGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERA GS linker;TLSCWLSQSVSSTYLAWYQQKPGQAPRLLIYDASSRAPGIPDRF C01_Rd4_6nM_C12 VL;SGSGSGTDFTLTISRLEPEDFAVYYCQQYSEWPLTFGQGTKVEIK CD8α hinge;TTTPAPRPPTPAPTIASQPLSLRPEACRpAAGGAVHTRGLDFAC CD8α TM domain;DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT 41BB ISD;TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLY CD3ζ ISDNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR (SEQ ID NO: 356)PC1C12- MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA CD8α signalpeptide; V3 ASGFTFSSYPMSWVRQAPGKGLEWVSAIGgSGGWSYYADSVK C01_Rd4_6nM_C12GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDSWG VH;QGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERA GS linker;TLSCWLSQSVSSTYLAWYQQKPGQAPRLLIYDASSRAPGIPDRF C01_Rd4_6nM_C12 VL;SGSGSGTDFTLTISRLEPEDFAVYYCQQYSEWPLTFGQGTKVEIK IgG1 hinge;EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVT CD8α TM domain;CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV 41BB ISD;VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP CD3ζ ISDQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR (SEQ IDNO: 357) COM22- MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA CD8α signalpeptide; V1 ASGFTFSSYAMNWVRQAPGKGLEWVSAISdSGGSRWYADSVCOMBO_Rd4_0.6nM_C22 KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRYWPMDIWG VH (SEQID NO: 92); QGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERA GS linker;TLSCRASVRVSSTYLAWYQQKPGQAPRLLMYDASIRATGIPDRF COMBO_Rd4_0.6nM_C22SGSGSGTDFTLTISRLEPEDFAVYYCQQYMKWPLTFGQGTKVEI VL (SEQ ID NO: 93);KGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRK FcγRIIIα hinge;KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS CD8α TM domain;ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK 41BB ISD;PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY CD3ζ ISDQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 358) COM22-MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA CD8α signal peptide; V2ASGFTFSSYAMNWVRQAPGKGLEWVSAISdSGGSRWYADSV COMBO_Rd4_0.6nM_C22KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRYWPMDIWG VH;QGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERA GS linker;TLSCRASVRVSSTYLAWYQQKPGQAPRLLMYDASIRATGIPDRF COMBO_Rd4_0.6nM_C22SGSGSGTDFTLTISRLEPEDFAVYYCQQYMKWPLTFGQGTKVEI VL;KTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA CD8α hinge;CDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPV CD8α TM domain;QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQ 41BB ISD;LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE CD3ζ ISDLQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPR (SEQ ID NO: 359)COM22- MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA CD8α signal peptide;V3 ASGFTFSSYAMNWVRQAPGKGLEWVSAISdSGGSRWYADSV COMBO_Rd4_0.6nM_C22KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRYWPMDIWG VH;QGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERA GS linker;TLSCRASVRVSSTYLAWYQQKPGQAPRLLMYDASIRATGIPDRF COMBO_Rd4_0.6nM_C22SGSGSGTDFTLTISRLEPEDFAVYYCQQYMKWPLTFGQGTKVEI VL;KEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEV IgG1 hinge;TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR CD8α TM domain;VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE 41BB ISD;PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE CD3ζ ISDNNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR (SEQID NO: 360) P6DY-V1 MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCACD8α signal peptide; ASGFTFGSYAMTWVRQAPGKGLEWVSAIDYSGGNTFYADSVKL3.PY/H2.DY VH (SEQ GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSPIASGMDY ID NO:25); WGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPG GS linker;ERATLSCRASQSVSSSYPSWYQQKPGQAPRLLIYGASSRATGIP L3.PY/L1.PS/P6E01DRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYPYPPSFTFGQGTK VL (SEQ ID NO: 18);VEIKGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRG FcγRIIIα hinge;RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFS CD8α TM domain;RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG 41BB ISD;GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD CD3ζ ISD GLYQGLSTATKDTYDALHMQ(SEQ ID NO: 361) P6DY-V2 MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCACD8α signal peptide; ASGFTFGSYAMTWVRQAPGKGLEWVSAIDYSGGNTFYADSVKL3.PY/H2.DY VH; GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSPIASGMDY GS linker;WGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPG L3.PY/L1.PS/P6E01ERATLSCRASQSVSSSYPSWYQQKPGQAPRLLIYGASSRATGIP VL;DRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYPYPPSFTFGQGTK CD8α hinge;VEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD CD8α TM domain;FACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRP 41BB ISD;VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQN CD3ζ ISDQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR (SEQ ID NO: 362)P6DY-V3 MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA CD8α signalpeptide; ASGFTFGSYAMTWVRQAPGKGLEWVSAIDYSGGNTFYADSVK L3.PY/H2.DY VH;GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSPIASGMDY GS linker;WGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPG L3.PY/L1.PS/P6E01ERATLSCRASQSVSSSYPSWYQQKPGQAPRLLIYGASSRATGIP VL;DRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYPYPPSFTFGQGTK IgG1 hinge;VEIKEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTP CD8α TM domain;EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST 41BB ISD;YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP CD3ζ ISDREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR (SEQID NO: 363) P6AP-V1 MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCACD8α signal peptide; ASGFTFGSYAMTWVRQAPGKGLEWVSAISGSGGNTFYADSVK P6AP-V1VH (SEQ ID GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSPIAAPMDY NO: 8);WGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPG GS linker;ERATLSCRASQLGSFYLAWYQQKPGQAPRLLIYGASSRATGIPD P6AP-V1 VL (SEQ IDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYNYPPSFTFGQGTKV NO: 80)EIKGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGR FcγRIIIα hinge;KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGcELRVKFSR CD8α TM domain;SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG 41BB ISD;KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL CD3ζ ISD YQGLSTATKDTYDALHMQA(SEQ ID NO: 364) P6AP-V2 MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCACD8α signal peptide; ASGFTFGSYAMTWVRQAPGKGLEWVSAISGSGGNTFYADSVKL1.LGF/L3.KW/H3.AP GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSPIAAPMDY VH;WGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPG GS linker;ERATLSCRASQLGSFYLAWYQQKPGQAPRLLIYGASSRATGIPD P6AP-V1 VL;RFSGSGSGTDFTLTISRLEPEDFAVYYCQHYNYPPSFTFGQGTKV CD8α hinge;EIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF CD8α TM domain;ACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPV 41BB ISD;QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQ CD3ζ ISDLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPR (SEQ ID NO: 365)P6AP-V3 MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCA CD8α signalpeptide; ASGFTFGSYAMTWVRQAPGKGLEWVSAISGSGGNTFYADSVK L1.LGF/L3.KW/H3.APGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSPIAAPMDY VH;WGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPG GS linker;ERATLSCRASQLGSFYLAWYQQKPGQAPRLLIYGASSRATGIPD P6AP-V1 VL;RFSGSGSGTDFTLTISRLEPEDFAVYYCQHYNYPPSFTFGQGTKV IgG1 hinge;EIKEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPE CD8α TM domain;VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY 41BB ISD;RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR CD3ζ ISDEPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR (SEQID NO: 366)

For the activity assays, T cells from thirteen healthy donors (Donors1-13) were obtained. Briefly, the T cells were purified from buffy-coatsamples and activated using CD3/CD28 beads. Cells were transientlytransfected with mRNAs encoding the different CAR molecules at D11/12after activation. CAR activity was assessed by measuring theirdegranulation capacity, the inteferon-γ (IFNγ) release, and thecytotoxic activity when co-cultured with (a) cells expressing BCMA(MM1S, KMS12BM, and L363), or (b) cells that do not express the BCMAprotein (K562). Also included for each assay were mock transfected Tcells (T cells in in buffer) to determine baseline activity of T cellsthat do not express a CAR.

CAR detection was done using a fusion protein in which the extracellulardomain of the human BCMA protein was fused to a mouse IgG1 derived Fcfragment. Binding of the CAR at the cell surface with the BCMA portionof the fusion protein was detected with anti-Fc PE-conjugated antibodyand analyzed by flow cytometry.

Materials and Methods

Primary T Cell Cultures

T cells were purified from Buffy coat samples provided by EFS(Etablissement Francais du Sang, Paris, France) using Ficoll gradientdensity medium (Ficoll Paque PLUS/GE Healthcare Life Sciences). The PBMClayer was recovered and T cells were purified using a commerciallyavailable T cell enrichment kit (Stem Cell Technologies). Purified Tcells were activated in X-Vivo™-15 medium (Lonza) supplemented with20ng/mL Human IL-2 (Miltenyi Biotech), 5% Human Serum (SeraLaboratories), and Dynabeads Human T activator CD3/CD28 at a bead:cellratio 1:1 (Life Technologies). After activation cells were grown andmaintained in X-Vivo™-15 medium (Lonza) supplemented with 20ng/mL HumanIL-2 (Miltenyi Biotec) and 5% Human Serum (Sera Laboratories)

CAR mRNA Transfection

Transfections were done at Day 4/5 or Day 11/12 after T cellpurification and activation. 5 millions of cells were transfected with15 μg of mRNA encoding the different CAR constructs. CAR mRNAs wereproduced using the mMESSAGE mMACHINE T7 Kit (Life Technologies) andpurified using RNeasy Mini Spin Columns (Qiagen). Transfections weredone using PulseAgile™ Cytopulse technology, by applying two 0.1 mSpulses at 3000V/cm followed by four 0.2 mS pulses at 325V/cm in 0.4 cmgap cuvettes in a final volume of 200 μl of “Cytoporation buffer T” (BTXHarvard Apparatus). Cells were immediately diluted in X-Vivo™-15 media(Lonza) and incubated at 37° C. with 5% CO₂. IL-2 (from Miltenyi Biotecwas added 2 h after electroporation at 20 ng/mL.

Degranulation Assay (CD107a Mobilization)

T cells were incubated in 96-well plates (50,000 cells/well), togetherwith an equal amount of cells expressing or not the BCMA protein.Co-cultures were maintained in a final volume of 100 μl of X-Vivo™-15medium (Lonza) for 6 hours at 37° C. with 5% CO₂. CD107a staining wasdone during cell stimulation, by the addition of a fluorescentanti-CD107a antibody (APC conjugated, from Miltenyi Biotec) at thebeginning of the co-culture, together with 1 μg/ml of anti-CD49d (BDPharmingen), 1 μg/ml of anti-CD28 (Miltenyi Biotec), and 1× Monensinsolution (eBioscience). After the 6h incubation period, cells werestained with a fixable viability dye (eFluor 780, from eBioscience) andfluorochrome-conjugated anti-CD8 (PE conjugated Miltenyi Biotec) andanalyzed by flow cytometry. The degranulation activity was determined asthe % of CD8+/CD107a+ cells, and by determining the mean fluorescenceintensity signal (MFI) for CD107a staining among CD8+ cells.Degranulation assays were carried out 24 h after mRNA transfection.Results are summarized in the Tables 6A-9H and 9A-9C below. In thetables, the second column (labeled “CAR-T cell”) indicates the BCMAspecific CAR being expressed in the transfected T cells.

CD107a expression on cells is a marker of antigen specific activation.The percent and MFI of CD107a on CD8 T cells expressing BCMA specificCARs increase when incubated with BCMA high (H929), medium (MM1S) andlow (KMS12BM, L363) expressing cells but not BCMA negative cells (K562and Daudi) (Tables 6A-9H and 9A-9C). CD107a expression levels did notincrease on mock transfected T cells contacted with BCMA. Thus, the BCMAspecific CAR-T cells are activated in the presence of BCMA-expressingcells but not in the presence of cells that do not express BCMA.

These results demonstrate that T cells expressing BCMA specific CARs areactivated when incubated with BCMA expressing cells, and that theactivation is antigen-specific.

IFN γ Release Assay

T cells were incubated in 96-well plates (50,000 cells/well), togetherwith (a) cells expressing BCMA (MM1S, KMS12BM, and L363) or (b) cellsthat do not express the BCMA protein (K562). Co-cultures were maintainedin a final volume of 100 μl of X-Vivo™-15 medium (Lonza) for 24 hours at37° C. with 5% CO₂. After this incubation period the plates werecentrifuged at 1500 rpm for 5 minutes and the supernatants wererecovered in a new plate. IFNγ detection in the cell culturesupernatants was done by ELISA assay (Human IFNγ Quantikine ELISA Kit,from R&D Systems). The IFNγ release assays were carried by starting thecell co-cultures 24 h after mRNA transfection. Results are summarized inthe Tables 8A-8D and 10 below.

As shown in Tables 8A-8D and 10, CD8 T cells expressing BCMA specificCARs produce IFNγ when incubated with either medium BCMA-expressingcells (MM1S) or low BCMA-expressing cells (KMS12BM, L363). In contrast,CD8 T cells expressing BCMA specific CARs produce negligible IFNγ whenincubated with BCMA negative cells (K562).

These results demonstrate that T cells expressing BCMA specific CARs areactivated when incubated with BCMA expressing cells, and that theactivation is antigen-specific.

Cytotoxicity Assay

T cells were incubated in 96-well plates (100,000 cells/well), togetherwith 10,000 target cells (expressing BCMA) and 10,000 control (BCMAneg)cells in the same well. Target and control cells were labelled withfluorescent intracellular dyes (CFSE or Cell Trace Violet, from LifeTechnologies) before co-culturing them with CAR+ T cells. Theco-cultures were incubated for 4 hours at 37° C. with 5% CO₂. After thisincubation period, cells were labelled with a fixable viability dye(eFluor 780, from eBioscience) and analyzed by flow cytometry. Viabilityof each cellular population (target cells or BCMAneg control cells) wasdetermined and the % of specific cell lysis was calculated. Cytotoxicityassays were carried out 48h after mRNA transfection. Results aresummarized in the Tables 7A-7H below. In the tables, the cytotoxicitydata are shown as percent viable cells, then calculated as a ratio oflive BCMA positive cells/live BCMA negative cells. Cell lysis iscalculated as 100—mock transfected T cells.

As shown in Tables 7A-7H, T cells expressing BCMA specific CARs exhibitkilling activity when incubated with either medium BCMA-expressing cells(MM1S) or low BCMA-expressing cells (L363). In contrast, CD8 T cellsexpressing BCMA specific CARs do not exhibit killing activity whenincubated with BCMA negative cells (K562).

In summary, T cells expressing the selected BCMA specific CARs shown inTable 5 are selectively activated upon contact with BCMA-expressingcells. While all versions of the BCMA specific CARs exhibitedBCMA-specific activiation, BCMA specific CARs comprising a CD8α hinge(v2) exhibited increased activation levels compared to BCMA specificCARs comprising a FcγRIIIα (v1) hinge or IgG1 (v3) hinge.

TABLE 6A Degranulation Assay Results, Donor 1 % MFI CD107a+ CD107a+ (inCD8+) Donor 1 mock T cells 410 2.45 transfected PMA/Iono 4038 76.1 Tcells MM1S 547 6.78 K562 610 7.55 P6DY v1 T cells 588 5.19 PMA/Iono 375875.1 MM1S 850 14.9 K562 829 9.76 v2 T cells 756 6.86 PMA/Iono 4103 75.5MM1S 3872 75.4 K562 1130 20.7 v3 T cells 707 7.71 PMA/Iono 4336 78.7MM1S 3665 72.6 K562 612 7.7 P6AP v1 T cells 604 4.61 PMA/Iono 3526 72.8MM1S 1847 46.4 K562 503 4.28 v2 T cells 1380 27.8 PMA/Iono 2504 58 MM1S5299 83.9 K562 949 14.6 v3 T cells 856 12.6 PMA/Iono 2500 58.9 MM1S 363873 K562 718 9.15

TABLE 6B Degranulation Assay Results, Donor 2 % MFI CD107a+ CD107a+ (inCD8+) Donor 2 mock T cells 270 1.66 transfected T PMA/Iono 3872 88.3cells MM1S 499 11 K562 492 8.78 P5A v1 T cells 423 7.2 PMA/Iono 603496.3 MM1S 2670 77.6 K562 648 16.6 v2 T cells 428 7.14 PMA/Iono 4420 90.7MM1S 5019 91.8 K562 620 13.8 v3 T cells 451 8.87 PMA/Iono 4835 93.2 MM1S4191 88.5 K562 607 14.1 P5A_C1 v1 T cells 315 4.12 PMA/Iono 3567 85.8MM1S 2193 68.6 K562 537 10.1 v2 T cells 413 7.46 PMA/Iono 4423 91.1 MM1S4575 90.6 K562 660 17.2 v3 T cells 429 7.82 PMA/Iono 4442 93.5 MM1S 371084.4 K562 597 13.9 P5A_C16 v1 T cells 424 7.95 PMA/Iono 4325 91.1 MM1S1858 61.6 K562 636 14.9 v2 T cells 401 5.69 PMA/Iono 3007 80 MM1S 422887.9 K562 696 17.6 v3 T cells 372 5.25 PMA/Iono 3611 86.6 MM1S 3372 83.6K562 476 7.72

TABLE 6C Degranulation Assay Results, Donor 3 % MFI CD107a+ CD107a+ (inCD8+) Donor 3 mock T cells 338 3.61 transfected T PMA/Iono 7111 98.1cells MM1S 464 9.44 K562 533 9.73 PC1 v1 T cells 454 6.67 PMA/Iono 522696.5 MM1S 2178 75.6 K562 753 22.3 v2 T cells 507 13 PMA/Iono 4743 95.2MM1S 759 25.5 K562 649 15.5 v3 T cells 463 6.84 PMA/Iono 7092 98.1 MM1S2857 87.2 K562 665 15 PC1C12 v1 T cells 373 3.35 PMA/Iono 6214 97.2 MM1S1960 68.2 K562 513 7.61 v2 T cells 579 11.5 PMA/Iono 6341 97.5 MM1S 447895.1 K562 680 15 v3 T cells 533 10.1 PMA/Iono 5785 97.4 MM1S 3739 91K562 648 13.2 COM22 v1 T cells 354 2.74 PMA/Iono 5894 96.7 MM1S 221976.1 K562 445 5.62 v2 T cells 401 6.52 PMA/Iono 5802 94.6 MM1S 2372 79.2K562 534 8.9 v3 T cells 501 10.4 PMA/Iono 6387 97.6 MM1S 2780 85.9 K562648 13.8

TABLE 6D Degranulation Assay Results, Donor 4 % MFI CD107a+ CD107a+ (inCD8+) Donor mock T cells 248 2.64 4 (v3 transfected PMA/Iono 5750 94.9only) T cells MM1S 363 8.89 K562 368 6.86 P5A T cells 335 3.82 PMA/Iono6025 93 MM1S 3150 86.7 K562 418 9.91 P5AC1 T cells 505 22.1 PMA/Iono6950 98.3 MM1S 2975 84.7 K562 575 23.3 P5AC16 T cells 368 6.2 PMA/Iono5775 97.7 MM1S 3675 86.8 K562 420 9.73 PC1 T cells 403 9.05 PMA/Iono6975 97.8 MM1S 4625 93 K562 543 15.8 PC1C12 T cells 485 12.9 PMA/Iono6400 96.5 MM1S 3575 90.4 K562 585 18.9 COM22 T cells 535 20.5 PMA/Iono7250 98.3 MM1S 3725 91.4 K562 533 16.9 P6DY T cells 313 3.08 PMA/Iono5125 94.3 MM1S 2435 79.9 K562 438 10.4 P6AP T cells 430 10.4 PMA/Iono6100 94.2 MM1S 3800 91.7 K562 478 14.6

TABLE 6E Degranulation Assay Results, Donor 5 % CD107a+ MFI (in CD8+)CD107a+ Donor CAR- L363 47 917 5 (v3 BCMA-P5A MM1S 65.3 1713 only) K5623.65 247 T cells 1.71 199 PMA/iono 98.6 4797 CAR- L363 50.6 1117 BCMA-MM1S 65.5 1753 P5AC1 K562 5.29 265 T cells 1.93 213 PMA/iono 99.1 5755CAR- L363 57.2 1392 BCMA- MM1S 73.9 2520 P5AC16 K562 4.13 273 T cells2.57 232 PMA/iono 98.1 5120 CAR- L363 71.9 2167 BCMA- MM1S 82.9 2987 PC1K562 4.5 316 T cells 2.47 273 PMA/iono 98.5 5556 CAR- L363 57.8 1492BCMA- MM1S 71.5 2094 PC1C12 K562 3.72 313 T cells 2.53 272 PMA/iono 98.24480 CAR- L363 61.3 1574 BCMA- MM1S 78.1 2602 COM22 K562 5.84 296 Tcells 5.26 284 PMA/iono 98.3 4434 CAR- L363 43.4 859 BCMA- MM1S 63.61624 P6DY K562 3.99 256 T cells 1.95 228 PMA/iono 98.1 4075 CAR- L36363.4 1745 BCMA- MM1S 77.8 2461 P6AP K562 4.81 310 T cells 4.74 300PMA/iono 98.9 32 mock L363 2.54 200 transfected MM1S 5.19 233 T cellsK562 4.02 201 T cells 1.95 192 PMA/iono 97.7 3216

TABLE 6F Degranulation Assay Results, Donor 6 % MFI CD107a+ CD107a+ (inCD8+) Donor 6 BCMA_BC30_v3 (18) Tcells alone 121 1.04 T cells PMA 525387.4 IONO T cells K562 230 3.21 T cells MM1S 1321 50.4 T cells L363 98641.8 CAR_BCMA_P5AC1_v2 Tcells alone 150 1.07 T cells PMA 4701 83.2 IONOT cells K562 256 5.5 T cells MM1S 2193 63.8 T cells L363 1400 50.9CAR_BCMA_P5AC1_v3 Tcells alone 166 0.96 T cells PMA 4518 80.2 IONO Tcells K562 301 6.87 T cells MM1S 1101 40.7 T cells L363 728 29.8CAR_BCMA_PC1_v3 Tcells alone 217 1.63 T cells PMA 4711 82.4 IONO T cellsK562 329 6.36 T cells MM1S 2083 60.3 T cells L363 1500 52.1CAR_BCMA_PC1C12_v2 Tcells alone 209 2.01 T cells PMA 5401 87.8 IONO Tcells K562 332 7.7 T cells MM1S 2588 68.4 T cells L363 1976 59.5CAR_BCMA_PC1C12_v3 Tcells alone 162 1.72 T cells PMA 5299 85.3 IONO Tcells K562 266 6.25 T cells MM1S 669 28.8 T cells L363 414 18.6CAR_BCMA_COM22_v3 Tcells alone 193 3.23 T cells PMA 4750 82.7 IONO Tcells K562 288 5.13 T cells MM1S 814 35.7 T cells L363 606 26.8CAR_BCMA_P6AP_v2 Tcells alone 359 9.69 T cells PMA 5521 87.4 IONO Tcells K562 327 7.69 T cells MM1S 2289 63.8 T cells L363 1876 56.9CAR_BCMA_P6AP_v3 Tcells alone 284 4.87 T cells PMA 4480 82.7 IONO Tcells K562 331 5.9 T cells MM1S 1409 46.9 T cells L363 926 35.3 mocktransfected T cells Tcells alone 184 0.92 T cells PMA 3955 78.6 IONO Tcells K562 278 3.58 T cells MM1S 393 4.7 T cells L363 190 1.12

TABLE 6G Degranulation Assay Results, Donor 7 % MFI CD107a+ CD107a+ (inCD8+) Donor 7 mock transfected T Tcells alone 68.3 1.55 cells T cellsPMA 3097 94.6 IONO T cells MM1S 118 7.15 T cells L363 90.3 2.63 T cellsK562 144 3.4 T cells Daudi 117 1.93 BCMA_BC30_v3 Tcells alone 69.7 2.69(18) T cells PMA 2864 94.9 IONO T cells MM1S 1630 68.9 T cells L363 52943.8 T cells K562 125 3.85 T cells Daudi 426 38.5 P5AC1_v2 Tcells alone111 3.67 T cells PMA 2859 95.6 IONO T cells MM1S 2305 71.5 T cells L363877 53.1 T cells K562 166 8.54 T cells Daudi 770 51.5 P5AC1_v3 Tcellsalone 70.8 1.04 T cells PMA 2740 94.6 IONO T cells MM1S 526 43.3 T cellsL363 209 20.4 T cells K562 118 8.32 T cells Daudi 450 35.9 PC1_v3 Tcellsalone 61 1.37 T cells PMA 2786 94.6 IONO T cells MM1S 1027 56.3 T cellsL363 314 29.9 T cells K562 140 12.1 T cells Daudi 536 39.6 PC1C12_v2Tcells alone 98 5.95 T cells PMA 3493 95.3 IONO T cells MM1S 1917 73.7 Tcells L363 939 56.2 T cells K562 192 11.5 T cells Daudi 1485 64.6PC1C12_v3 Tcells alone 84.2 2.28 T cells PMA 3017 95.2 IONO T cells MM1S342 28.2 T cells L363 145 8.72 T cells K562 186 7.53 T cells Daudi 22311.8 COM22_v3 Tcells alone 93.6 5.32 T cells PMA 2989 96.3 IONO T cellsMM1S 540 40 T cells L363 154 12.5 T cells K562 138 8.29 T cells Daudi93.5 3.99 P6AP_v2 Tcells alone 164 13.7 T cells PMA 3303 95.9 IONO Tcells MM1S 2755 76 T cells L363 859 50.3 T cells K562 287 15.8 T cellsDaudi 1263 58.2 P6AP_v3 Tcells alone 114 10.5 T cells PMA 3084 94.5 IONOT cells MM1S 849 51.6 T cells L363 380 30.9 T cells K562 211 8.46 Tcells Daudi 678 42.7

TABLE 6H Degranulation Assay Results, Donor 8 % MFI CD107a+ CD107a+ (inCD8+) Donor 8 mock transfected T Tcells alone 154 0.67 cells T cells PMA3777 66.2 IONO T cells MM1S 229 2.16 T cells L363 166 1.51 T cells K562220 2.08 BCMA_BC30_v3 Tcells alone 210 1.05 (18) T cells PMA 4302 70.6IONO T cells MM1S 1661 42 T cells L363 1049 26.5 T cells K562 262 3.46P5AC1_v2 Tcells alone 207 0.86 T cells PMA 4298 71.5 IONO T cells MM1S1648 40.8 T cells L363 1099 26.5 T cells K562 232 1.72 P5AC1_v3 Tcellsalone 187 0.84 T cells PMA 3989 68.8 IONO T cells MM1S 766 21.2 T cellsL363 521 14.2 T cells K562 258 2.05 PC1_v3 Tcells alone 242 1.23 T cellsPMA 4256 70.6 IONO T cells MM1S 1046 23.1 T cells L363 1183 27.4 T cellsK562 283 2.97 PC1C12_v2 Tcells alone 257 1.87 T cells PMA 3487 60.2 IONOT cells MM1S 2463 51.2 T cells L363 1657 35.4 T cells K562 314 4.05PC1C12_v3 Tcells alone 166 0.86 T cells PMA 4238 69.1 IONO T cells MM1S641 17.3 T cells L363 507 14.2 T cells K562 296 3.52 COM22_v3 Tcellsalone 283 2.55 T cells PMA 4800 75.9 IONO T cells MM1S 1035 27.9 T cellsL363 704 22.7 T cells K562 334 4.82 P6AP_v2 Tcells alone 545 8.33 Tcells PMA 4362 68.6 IONO T cells MM1S 2273 46.7 T cells L363 1671 34.7 Tcells K562 629 9.71 P6AP_v3 Tcells alone 360 3.87 T cells PMA 3584 61.5IONO T cells MM1S 1553 34.5 T cells L363 1045 23 T cells K562 595 7.4

TABLE 7A Cytotoxicity Data, Donor 6 Viability (mean) CAR L363 K562 MM1SK562 Donor 6 BC30_v3 22.93 89.90 16.30 88.43 P5AC1_v2 27.27 90.07 21.4790.17 P5AC1_v3 36.03 89.30 19.80 88.50 PC1_v3 19.03 88.23 13.57 87.50PC1C12_v2 19.60 86.13 14.67 84.67 PC1C12_v3 55.50 89.33 41.33 88.67COM22_v3 42.00 90.33 25.67 88.30 P6AP_v2 29.40 80.27 21.07 82.10 P6AP_v348.53 85.20 25.57 81.30 mock 90.90 88.20 91.77 86.30 transfected T cells

TABLE 7B Cytotoxicity Data, Donor 6 Ratio to Mock BCMA+/BCMA−transfected T cells Cell lysis CAR L363 MM1S L363 MM1S L363 MM1S Donor 6BC30_v3 25.51 18.43 0.24752108 0.17333946 75.2 82.7 P5AC1_v2 30.27 23.810.29374647 0.22389502 70.6 77.6 P5AC1_v3 40.35 22.37 0.391523360.21040098 60.8 79.0 PC1_v3 21.57 15.50 0.2093085 0.14581122 79.1 85.4PC1C12_v2 22.76 17.32 0.22079514 0.1629089 77.9 83.7 PC1C12_v3 62.1346.62 0.60281513 0.4383953 39.7 56.2 COM22_v3 46.49 29.07 0.451134410.27335977 54.9 72.7 P6AP_v2 36.63 25.66 0.35539949 0.24131177 64.5 75.9P6AP_v3 56.96 31.45 0.55272006 0.29573955 44.7 70.4 mock transfected Tcells 103.06 106.33 1 1 0.0 0.0

TABLE 7C Cytotoxicity Data, Donor 7 Viability (mean) CAR L363 K562 MM1SK562 Donor 7 mock 92.53 92.80 90.70 92.33 transfected T cells BC30_v346.00 90.40 34.00 89.83 P5AC1_v2 50.50 90.73 35.17 89.40 P5AC1_v3 60.2089.97 43.03 89.53 PC1_v3 49.43 89.67 37.33 88.97 PC1C12_v2 40.23 88.5022.53 87.53 PC1C12_v3 81.03 91.30 71.70 89.83 COM22_v3 67.87 90.00 52.9789.20 P6AP_v2 57.33 89.93 32.87 87.10 P6AP_v3 66.37 91.60 46.35 94.00

TABLE 7D Cytotoxicity Data, Donor 7 Ratio to Mock BCMA+/BCMA−transfected T cells Cell lysis CAR L363 MM1S L363 MM1S L363 MM1S Donor 7mock transfected T cells 99.71 98.23 1 1 0.0 0.0 BC30_v3 50.88 37.850.51031598 0.38529434 49.0 61.5 P5AC1_v2 55.66 39.34 0.558180010.40044688 44.2 60.0 P5AC1_v3 66.91 48.06 0.67106507 0.48929577 32.951.1 PC1_v3 55.13 41.96 0.55288988 0.4271896 44.7 57.3 PC1C12_v2 45.4625.74 0.45592406 0.26206149 54.4 73.8 PC1C12_v3 88.76 79.81 0.890107980.81251777 11.0 18.7 COM22_v3 75.41 59.38 0.7562472 0.60448985 24.4 39.6P6AP_v2 63.75 37.73 0.63934647 0.38413929 36.1 61.6 P6AP_v3 72.45 49.310.7266149 0.50196463 27.3 49.8

TABLE 7E Cytotoxicity Data, Donor 8 Viability (mean) CAR L363 K562 MM1SK562 Donor 8 mock transfected 93.97 91.13 95.97 88.07 T cells BC30_v367.97 86.80 46.40 78.87 P5AC1_v2 69.80 85.37 47.13 79.17 P5AC1_v3 77.9088.77 62.70 84.40 PC1_v3 61.67 86.60 41.67 78.97 PC1C12_v2 62.43 85.2735.27 78.20 PC1C12_v3 85.17 85.27 78.87 77.77 COM22_v3 76.70 87.87 56.4084.50 P6AP_v2 77.23 84.90 61.47 83.47 P6AP_v3 83.23 85.67 72.57 84.63cell lines 95.20 94.97 96.97 94.20

TABLE 7F Cytotoxicity Data, Donor 8 Ratio to Mock BCMA+/BCMA−transfected T cells Cell lysis CAR L363 MM1S L363 MM1S L363 MM1S Donor 8mock transfected T cells 1.03 0.95 1 1 0.0 0.0 BC30_v3 0.78 0.590.75941589 0.61953757 24.1 38.0 P5AC1_v2 0.82 0.60 0.79299515 0.6269442920.7 37.3 P5AC1_v3 0.88 0.74 0.85112036 0.78229085 14.9 21.8 PC1_v3 0.710.53 0.69061501 0.5556331 30.9 44.4 PC1C12_v2 0.73 0.45 0.71013460.47489852 29.0 52.5 PC1C12_v3 1.00 1.01 0.96871004 1.06793091 3.1 −6.8COM22_v3 0.87 0.67 0.84659295 0.70285469 15.3 29.7 P6AP_v2 0.91 0.740.88226799 0.77547847 11.8 22.5 P6AP_v3 0.97 0.86 0.94229927 0.90289845.8 9.7 cell lines 1.00 1.03 0.97223038 1.08396365 2.8 −8.4

TABLE 7G Cytotoxicity Data, Donor 9 Viability (mean) CAR L363 K562 MM1SK562 Donor 9 mock transfected 86.3 87.8 69.6 86.5 T cells BC30_v3 27.186.6 16.0 86.6 P5AC1_v2 31.9 87.9 21.0 87.2 P5AC1_v3 46.9 85.1 36.3 84.0PC1_v3 27.8 85.3 25.4 85.0 PC1C12_v2 29.3 88.7 15.0 86.0 COM22_v3 49.088.8 35.7 87.5 P6AP_v2 41.4 85.7 22.8 84.0 P6AP_v3 56.4 84.3 44.9 84.4Cell lines 92.3 91.7 83.5 91.8

TABLE 7H Cytotoxicity Data, Donor 9 Ratio to Mock BCMA+/BCMA−transfected T cells Cell lysis CAR L363 MM1S L363 MM1S L363 MM1S Donor 9mock transfected T cells 0.98216319 0.80469954 1 1 0 0 BC30_v30.31331794 0.18444359 0.31900802 0.22920802 68.10 77.08 P5AC1_v20.3631539 0.24111578 0.36974905 0.29963455 63.03 70.04 P5AC1_v30.55133229 0.43231441 0.56134489 0.53723706 43.87 46.28 PC1_v30.32551778 0.29831439 0.33142942 0.37071525 66.86 62.93 PC1C12_v20.3298272 0.17473847 0.3358171 0.21714748 66.42 78.29 COM22_v30.55159475 0.40746382 0.56161212 0.50635523 43.84 49.36 P6AP_v20.48289269 0.27092424 0.49166238 0.33667751 50.83 66.33 P6AP_v30.66903915 0.53199052 0.68118939 0.66110454 31.88 33.89 Cell lines1.00690909 0.90889292 1.02519531 1.1294811 −2.52 −12.95

TABLE 8A IFNγ Production (pg/mL), Donor 6 Donor 6 CAR pg/ml mocktransfected T cells Tcells alone 155.1 BCMA_BC30_v3 (18) 654.71 P5AC1_v2174.035 P5AC1_v3 61.215 PC1_v3 255.045 PC1C12_v2 481.595 PC1C12_v3463.08 COM22_v3 2996.305 P6AP_v2 1294.055 P6AP_v3 500.435 mocktransfected T cells T cells PMA IONO 81654.2 BCMA_BC30_v3 (18) 49368.7P5AC1_v2 49102.7 P5AC1_v3 66837.7 PC1_v3 70798.2 PC1C12_v2 56402.2PC1C12_v3 121954.7 COM22_v3 125878.7 P6AP_v2 73577.2 P6AP_v3 51242.7mock transfected T cells T cells K562 −83.215 BCMA_BC30_v3 (18) 265.565P5AC1_v2 −10.05 P5AC1_v3 36.475 PC1_v3 −74.04 PC1C12_v2 344.72 PC1C12_v3583.99 COM22_v3 610.97 P6AP_v2 40.66 P6AP_v3 36.775 mock transfected Tcells T cells MM1S 660.33 BCMA_BC30_v3 (18) 8004.42 P5AC1_v2 5667.72P5AC1_v3 2619.735 PC1_v3 6152.67 PC1C12_v2 8526.27 PC1C12_v3 1405.945COM22_v3 3330.27 P6AP_v2 5436.27 P6AP_v3 3881.115 mock transfected Tcells T cells L363 1287.38 BCMA_BC30_v3 (18) 6363.72 P5AC1_v2 3116.725P5AC1_v3 2720.52 PC1_v3 6661.97 PC1C12_v2 9478.72 PC1C12_v3 1707.885COM22_v3 2397.83 P6AP_v2 5911.97 P6AP_v3 3470.38

TABLE 8B IFNγ Production (pg/mL), Donor 7 Donor 7 CAR pg/ml mocktransfected T cells Tcells alone −3.1 BCMA_BC30_v3 (18) 64.1 P5AC1_v2−18.0 P5AC1_v3 −73.0 PC1_v3 6.1 PC1C12_v2 156.5 PC1C12_v3 100.1 COM22_v3182.9 P6AP_v2 564.7 P6AP_v3 107.0 mock transfected T cells T cells PMAIONO 44970.8 BCMA_BC30_v3 (18) 32725.3 P5AC1_v2 27476.6 P5AC1_v3 13100.5PC1_v3 40824.4 PC1C12_v2 39884.0 PC1C12_v3 30245.2 COM22_v3 62690.4P6AP_v2 69923.2 P6AP_v3 88578.4 mock transfected T cells T cells MM1S29.9 BCMA_BC30_v3 (18) 4662.6 P5AC1_v2 3420.3 P5AC1_v3 1173.7 PC1_v32478.5 PC1C12_v2 5314.6 PC1C12_v3 809.9 COM22_v3 1344.6 P6AP_v2 3020.3P6AP_v3 2166.7 mock transfected T cells T cells L363 15.6 BCMA_BC30_v3(18) 2360.2 P5AC1_v2 2576.3 P5AC1_v3 582.7 PC1_v3 1723.3 PC1C12_v22962.9 PC1C12_v3 136.6 COM22_v3 467.4 P6AP_v2 2081.4 P6AP_v3 1119.0 mocktransfected T cells T cells K562 −80.5 BCMA_BC30_v3 (18) −127.2 P5AC1_v2−124.4 P5AC1_v3 −47.9 PC1_v3 −93.6 PC1C12_v2 21.8 PC1C12_v3 −55.4COM22_v3 −36.1 P6AP_v2 83.8 P6AP_v3 83.8 mock transfected T cells 335.1BCMA_BC30_v3 (18) T cells Daudi 7794.8 P5AC1_v2 8093.7 P5AC1_v3 3870.6PC1_v3 6068.9 PC1C12_v2 10190.2 PC1C12_v3 1638.8 COM22_v3 4287.6 P6AP_v26971.6 P6AP_v3 5280.0

TABLE 8C IFN-γ Production (pg/mL), Donor 8 Donor 8 CAR pg/ml mocktransfected T cells Tcells alone −697.44 BCMA_BC30_v3 (18) −660.92P5AC1_v2 −603.38 P5AC1_v3 −543.44 PC1_v3 −552.22 PC1C12_v2 −399.26PC1C12_v3 −652.73 COM22_v3 −530.09 P6AP_v2 17.24 P6AP_v3 −289.82 mocktransfected T cells T cells PMA IONO 37206.73 BCMA_BC30_v3 (18) 53311.73P5AC1_v2 57732.14 P5AC1_v3 52577.56 PC1_v3 48925.48 PC1C12_v2 38310.06PC1C12_v3 71881.73 COM22_v3 61941.73 P6AP_v2 82339.64 P6AP_v3 63337.14mock transfected T cells T cells MM1S −684.65 BCMA_BC30_v3 (18) 2976.34P5AC1_v2 2727.71 P5AC1_v3 769.05 PC1_v3 2682.98 PC1C12_v2 5019.05PC1C12_v3 −198.04 COM22_v3 1155.19 P6AP_v2 2945.65 P6AP_v3 671.21 mocktransfected T cells T cells L363 −664.74 BCMA_BC30_v3 (18) 2934.77P5AC1_v2 2342.50 P5AC1_v3 579.85 PC1_v3 2232.65 PC1C12_v2 3676.59PC1C12_v3 −303.86 COM22_v3 695.72 P6AP_v2 1612.74 P6AP_v3 311.07 mocktransfected T cells T cells K562 −672.42 BCMA_BC30_v3 (18) −583.71P5AC1_v2 −631.02 P5AC1_v3 −650.83 PC1_v3 −615.50 PC1C12_v2 −501.18PC1C12_v3 −615.17 COM22_v3 −596.02 P6AP_v2 −393.94 P6AP_v3 −476.71

TABLE 8D IFN-γ Production (pg/mL), Donor 9 Donor 9 CAR pg/ml mocktransfected T cells Tcells alone 93.2 BCMA_BC30_v3 (18) 1225.2 P5AC1_v21344.5 P5AC1_v3 632.3 PC1_v3 2745.7 PC1C12_v2 48.1 COM22_v3 2656.5P6AP_v2 566.5 P6AP_v3 −335.8 mock transfected T cells T cells PMA IONO12505.8 BCMA_BC30_v3 (18) 12312.2 P5AC1_v2 10607.5 P5AC1_v3 12014.7PC1_v3 12829.9 PC1C12_v2 13829.5 COM22_v3 13489.5 P6AP_v2 13182.1P6AP_v3 13506.3 mock transfected T cells T cells MM1S 1006.4BCMA_BC30_v3 (18) 2376.8 P5AC1_v2 −359.5 P5AC1_v3 97.8 PC1_v3 290.1PC1C12_v2 752.7 COM22_v3 −601.0 P6AP_v2 −304.1 P6AP_v3 −394.9 mocktransfected T cells T cells L363 −228.2 BCMA_BC30_v3 (18) 3000.2P5AC1_v2 2314.0 P5AC1_v3 1646.4 PC1_v3 −15.4 PC1C12_v2 2796.5 COM22_v3320.6 P6AP_v2 −163.0 P6AP_v3 −233.9 mock transfected T cells T cellsK562 −227.9 BCMA_BC30_v3 (18) 2027.5 P5AC1_v2 3928.4 P5AC1_v3 300.2PC1_v3 74.9 PC1C12_v2 1835.7 COM22_v3 45.0 P6AP_v2 51.4 P6AP_v3 158.3

TABLE 9A Degranulation Assay Results, Donor 10 % MFI CD107a+ Donor 10CD107a+ (in CD8+) LT alone mock transfected T cells 82.2 1.95 26859P5AC1-V2 83.8 1.47 26868 PC1C12-V2 94.2 3.21 26871 COM22-V2 107 5.96 PMAIono mock transfected T cells 5933 99 26859 P5AC1-V2 5863 99 26868PC1C12-V2 6366 99.4 26871 COM22-V2 6149 99 MM1S mock transfected T cells211 16.5 26859 P5AC1-V2 1377 74.4 26868 PC1C12-V2 1760 79.1 26871COM22-V2 1470 76.5 H929 mock transfected T cells 141 6.09 26859 P5AC1-V21026 65.4 26868 PC1C12-V2 1262 71.1 26871 COM22-V2 784 59.2 L363 mocktransfected T cells 153 6.48 26859 P5AC1-V2 793 60.1 26868 PC1C12-V21054 67.3 26871 COM22-V2 827 61.7 MM1S GFP LUC mock transfected T cells187 9.88 26859 P5AC1-V2 1228 70.5 26868 PC1C12-V2 1476 74.9 26871COM22-V2 1095 68.5 H929 GFP LUC mock transfected T cells 153 9.48 26859P5AC1-V2 1648 77.8 26868 PC1C12-V2 1960 84 26871 COM22-V2 1029 69.4 L363GFP LUC mock transfected T cells 104 3.06 26859 P5AC1-V2 753 60.7 26868PC1C12-V2 873 64.6 26871 COM22-V2 766 61.1 KMS12BM GFP LUC mocktransfected T cells 91.3 2.67 26859 P5AC1-V2 945 67.2 26868 PC1C12-V21192 71.2 26871 COM22-V2 961 67.2 K562 mock transfected T cells 127 6.0626859 P5AC1-V2 136 9.1 26868 PC1C12-V2 119 9.49 26871 COM22-V2 135 9.55

TABLE 9B Degranulation Assay Results, Donor 11 % MFI CD107a+ Donor 11CD107a+ (in CD8+) LT alone mock transfected T cells 69.9 0.57 26859P5AC1-V2 68.3 0.62 26868 PC1C12-V2 67.2 0.88 26871 COM22-V2 80.9 3.95PMA Iono mock transfected T cells 5511 91.7 26859 P5AC1-V2 5360 97.426868 PC1C12-V2 4741 96.1 26871 COM22-V2 5066 95.7 KMS12BM GFP LUC mocktransfected T cells 77.8 1.81 26859 P5AC1-V2 1304 68.3 26868 PC1C12-V2650 45.5 26871 COM22-V2 986 62.5 H929 GFP LUC mock transfected T cells73 1.04 26859 P5AC1-V2 738 49.6 26868 PC1C12-V2 428 30.9 26871 COM22-V2468 35.5 MM1S mock transfected T cells 121 2.67 26859 P5AC1-V2 854 5226868 PC1C12-V2 399 26.4 26871 COM22-V2 486 33.4 K562 mock transfected Tcells 125 3.08 26859 P5AC1-V2 140 3.35 26868 PC1C12-V2 123 1.84 26871COM22-V2 161 4.11

TABLE 9C Degranulation Assay Results, Donor 12 % MFI CD107a+ Donor 11CD107a+ (in CD8+) LT alone mock transfected T cells 69.9 0.57 26859P5AC1-V2 68.3 0.62 26868 PC1C12-V2 67.2 0.88 26871 COM22-V2 80.9 3.95PMA Iono mock transfected T cells 5511 91.7 26859 P5AC1-V2 5360 97.426868 PC1C12-V2 4741 96.1 26871 COM22-V2 5066 95.7 KMS12BM GFP LUC mocktransfected T cells 77.8 1.81 26859 P5AC1-V2 1304 68.3 26868 PC1C12-V2650 45.5 26871 COM22-V2 986 62.5 H929 GFP LUC mock transfected T cells73 1.04 26859 P5AC1-V2 738 49.6 26868 PC1C12-V2 428 30.9 26871 COM22-V2468 35.5 MM1S mock transfected T cells 121 2.67 26859 P5AC1-V2 854 5226868 PC1C12-V2 399 26.4 26871 COM22-V2 486 33.4 K562 mock transfected Tcells 125 3.08 26859 P5AC1-V2 140 3.35 26868 PC1C12-V2 123 1.84 26871COM22-V2 161 4.11

TABLE 10 IFN gamma release assay results, Donor 10 Donor 10 mocktransfected T cells T cells alone 871.8 pg/mL pCLS26859CAR_BCMA_P5AC1_v2 1466.2 pg/mL pCLS26868 CAR_BCMA_PC1C12_v2 1172.2 pg/mLpCLS26871 CAR_BCMA_COM22_v2 1873.1 pg/mL mock transfected T cells MM1SLucGFP 1436.5 pg/mL pCLS26859 CAR_BCMA_P5AC1_v2 12208.4 pg/mL pCLS26868CAR_BCMA_PC1C12_v2 13695.3 pg/mL pCLS26871 CAR_BCMA_COM22_v2 10784.1pg/mL mock transfected T cells MM1S 5329.0 pg/mL pCLS26859CAR_BCMA_P5AC1_v2 6060.3 pg/mL pCLS26868 CAR_BCMA_PC1C12_v2 6776.1 pg/mLpCLS26871 CAR_BCMA_COM22_v2 7827.0 pg/mL mock transfected T cells H929LucGFP 754.2 pg/mL pCLS26859 CAR_BCMA_P5AC1_v2 16589.9 pg/mL pCLS26868CAR_BCMA_PC1C12_v2 15989.7 pg/mL pCLS26871 CAR_BCMA_COM22_v2 14410.4pg/mL mock transfected T cells H929 809.8 pg/mL pCLS26859CAR_BCMA_P5AC1_v2 18072.7 pg/mL pCLS26868 CAR_BCMA_PC1C12_v2 17948.1pg/mL pCLS26871 CAR_BCMA_COM22_v2 14437.3 pg/mL mock transfected T cellsL363 LucGFP 1184.5 pg/mL pCLS26859 CAR_BCMA_P5AC1_v2 11556.9 pg/mLpCLS26868 CAR_BCMA_PC1C12_v2 13254.5 pg/mL pCLS26871 CAR_BCMA_COM22_v211384.1 pg/mL mock transfected T cells L363 1777.3 pg/mL pCLS26859CAR_BCMA_P5AC1_v2 15685.1 pg/mL pCLS26868 CAR_BCMA_PC1C12_v2 14929.1pg/mL pCLS26871 CAR_BCMA_COM22_v2 14995.7 pg/mL mock transfected T cellsL363 LucGFP 1184.5 pg/mL pCLS26859 CAR_BCMA_P5AC1_v2 11556.9 pg/mLpCLS26868 CAR_BCMA_PC1C12_v2 13254.5 pg/mL pCLS26871 CAR_BCMA_COM22_v211384.1 pg/mL mock transfected T cells KMS12BM LucGFP 1283.2 pg/mLpCLS26859 CAR_BCMA_P5AC1_v2 9073.3 pg/mL pCLS26868 CAR_BCMA_PC1C12_v210060.6 pg/mL pCLS26871 CAR_BCMA_COM22_v2 10687.2 pg/mL mock transfectedT cells K562 691.6 pg/mL pCLS26859 CAR_BCMA_P5AC1_v2 684.1 pg/mLpCLS26868 CAR_BCMA_PC1C12_v2 904.2 pg/mL pCLS26871 CAR BCMA COM22 v2969.0 pg/mL

Example 3 BCMA Specific CAR-T Cells Induce Tumor Regression in MM1.STumor Model

This example illustrates treatment of tumors with BCMA specific CAR-Tcells using the MM1.S tumor model.

In vivo efficacy study of BCMA specific CAR-T cells was performed withMM1.S, expressing luciferase and GFP, orthotopic model. Five millionMM1.S Luc2AGFP cells were injected intravenously through the tail veininto 6-8 weeks old female Nod/Scid/IL2Rg−/−(NSG) animals.Intraperitoneal injection of D-luciferin (Regis Technologies, MortonGrove, Ill.) (200 uL per animal at 15 mg/mL), followed by anesthesiawith isofluorane and subsequent whole body bioluminescence imaging (BLI)enable monitoring of tumor burden. Bioluminescent signals emitted by theinteraction between luciferase expressed by the tumor cells andluciferin were captured by imaging using an IVIS Spectrum CT (PerkinElmer, Mass.) and quantified as total flux (photons/sec) using LivingImage 4.4 (Caliper Life Sciences, Alameda, Calif.).

Three different BCMA specific CAR-T cells were used in this study: Tcells expressing the BCMA specific CAR contructs P5AC1-V2, PC1C12-V2, orCOM22-V2 (see, Table 5 above). Non-transduced control T cells were usedas the negative control. All T cells were engineered to be TCRαdeficient.

When the total flux reached an average of 45E6 for all animals (day 20post tumor implant), the animals were randomized into four groups. Asingle dose of human either BCMA specific CAR-T cells or non-transducedcontrol T cells was administered through bolus tail vein injection.Animals were terminated when they exhibit hindlimb paralysis or a 20%loss of body weight, an endpoint for MM1.S orthotopic models.

Results of this study are summarized in FIG. 1. In FIG. 1, total flux[p/s] represents tumor progression. Treatment with BCMA specific CAR-Tcells (triangles, diamonds, squares) resulted in lower total flux ascompared to the negative control (circles). Thus, treatment with BCMAspecific CAR-T cells inhibited tumor progression as compared to thenegative control.

These results demonstrate BCMA specific CAR-T cells are effective toinduce tumor regression.

Example 4 Treatment of Multiple Myeloma with BCMA Specific CAR-T Cells

This example illustrates treatment of multiple myeloma with BCMAspecific CAR-T cells using the Molp8 orthotopic model.

In vivo efficacy study of BCMA specific CAR-T cells was performed withMolp8, expressing luciferase and GFP, orthotopic model. Two millionMolp8 Luc2AGFP cells were injected intravenously through the tail veininto 6-8 weeks old female NSG animals. Intraperitoneal injection ofD-luciferin (Regis Technologies, Morton Grove, Ill.) (200 uL per animalat 15 mg/mL), followed by anesthesia with isofluorane and subsequentwhole body bioluminescence imaging (BLI) enable monitoring of tumorburden. Bioluminescent signals emitted by the interaction betweenluciferase expressed by the tumor cells and luciferin were captured byimaging using an IVIS Spectrum CT (Perkin Elmer, Mass.) and quantifiedas total flux (photons/sec) using Living Image 4.4 (Caliper LifeSciences, Alameda, Calif.).

When the total flux reached an average of 30E6 for all animals (day 8post tumor implant), the animals were randomized into three groups. Eachgroup was administered one of the following cells: 1) non-transduced Tcells TCR KO (“TCR KO”) used as a control, 2) BCMA specific CAR-T cellsexpressing P5AC1-V2.1 (“P5AC1 V2 R2 TCR KO”), or 3) BCMA specific CAR-Tcells expressing P5AC1-V2 and the RQR8 suicide polypeptide (“P5AC1 V2RQR8 TCR KO”). All of cells 1-3 are TCRα deficient. The BCMA specificCAR-T cells were prepared as described in example above. BCMA specificCAR constructs P5AC1-V2.1 and P5AC1-V2 are shown in Table 5 above. Asingle dose of 3 million control (TCR KO) or BCMA specific CAR-T (P5AC1V2 R2 TCR KO or P5AC1 V2 RQR8 TCR KO) cells were administered throughbolus tail vein injection. Animals were terminated when they lose morethan 15% of total body weight, an endpoint for Molp8 orthotopic models.

Results from the study are summarized in FIG. 2. A single dose of 3million P5AC1 R2 TCRKO BCMA specific CAR-T cells (squares) or P5AC1 RQR8TCRKO CAR-T cells (triangles) BCMA specific CAR-T cells resulted inlower total flux from days 10-35 post tumor implant as compared to thenegative control (circles) (FIG. 2). Thus, treatment with BCMA specificCAR-T cells inhibited tumor progression as compared to the negativecontrol.

These results demonstrate BCMA specific CAR-T cells are effective toinhibit tumor progression.

Example 5 Treatment of Multiple Myeloma with BCMA Specific CAR-T Cells

This example illustrates the therapeutic activity of BCMA specific CAR-Tcells in orthotopic mouse models of multiple myeloma.

Two humanized mouse models were used to evaluate the efficacy of BCMAspecific CAR-T cells against human myeloma cell lines expressing BCMA.Six (6) to eight (8) week old female Nod/Scid IL2rg−/− (NSG) mice werepurchased from the Jackson Laboratories. All animals were housed in apathogen free vivarium facility at Rinat and experiments were conductedaccording to the protocols in accordance with the Institutional AnimalCare and Use Committee (IACUC) guidelines.

The MM1.S and Molp-8 cell lines were purchased from the American TypeCulture Collection (ATCC.org) and the Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH (DSMZ.de). Cell lines wereengineered to express a Luc-GFP fusion protein using lentiviralparticles (amsbio). Cells were cultured in RPMI 1640 medium withL-glutamine supplemented with either 10% fetal calf serum for MM1.S orwith 20% FCS for Molp-8 cells at 37° C. in 5% carbon dioxide (CO₂).Cells growing in an exponential growth phase were harvested and used fortumor inoculation.

Therapeutic BCMA specific CAR-T cells were produced as described.Healthy human donor cells, peripheral blood mononuclear cells (PBMC) orpurified pan-T cells, are activated and transduced with lentiviralparticles encoding a BCMA specific CAR and RQR8 driven by EF-1apromoter. Three different BCMA specific CARs were used in this study:P5AC1-V2, PC1C12-V2 and COM22-V2 (see, Table 5 above). T cells were geneedited for deletion of the TCRα gene. Cells were cultured for 14 to 17days and then cryopreserved in 90% FCS/10% DMSO. For T cell injection, Tcells were rapidly thawed in a 37° C. waterbath and washed twice withRPMI 1640 medium containing 25mM Hepes. Cells were injected in 0.2 mlRPMI 1640 with 25 mM Hepes into the tail vein of tumor-bearing animals.

NSG mice were irradiated with 1 Gy total body irradiation (RAD SourceTechnologies) one day prior to tumor cell inoculation. 5×10⁶MM1.S/Luc2-EGFP cells or 2×10⁶ Molp-8/Luc2-EGFP cells were injected intothe tail vein in 0.1 ml of phosphate-buffered saline (PBS). Tumor burdenwas measured twice weekly using bioluminescence imaging. Mice wereinjected with 3 ug D-Luciferin dissolved in 0.2 ml PBS and anesthetizedusing isofluorane. 7 minutes after injection animals were imaged using aPerkin Elmer IVIS Spectrum camera system. The total body luminescencewith the exception of the mouse tail was measured and tumor burden isreported as total flux (photons per second). Tumors were allowed toestablish until exponential growth occurred. Animals were randomizedinto treatment groups based on total flux and treated with BCMA specificCAR-T cells or untransduced control T cells from the same donor. Theeffect of CAR-T treatment was assessed twice weekly usingbioluminescence imaging and body weight measurements. The study endpointwas reached when the first animal exhibited end-stage disease asindicated by body weight loss (>20% of initial body weight), hindlegparalysis, or other signs of animal distress. Statistical analysis wasperformed using GraphPad Prism 6. Repeated measures one-way ANOVA withTukey's correction was used to compare anti-tumor efficacy between allgroups. P<0.05 was considered significant.

Results are summarized in Table 11 (MM1.S) and Table 12 (Molp-8) below(log₁₀ values of total flux in photons per second+/−SEM). A suboptimalCAR-T cell dose was used to compare BCMA specific CAR-T cells havingdifferent scFvs. The BCMA specific CAR-T cell groups are P5AC1-V2,PC1C12-V2 and COM22-V2 (see, Table 5 above). In the MM1.S model, 3.5×10⁶CAR-expressing T cells were injected on day 17 after tumor implantation.In the Molp8 model, 4×10⁶ CAR-expressing T cells were injected on day 7after tumor implantation. Transduction efficiencies ranged from 19% to29% for BCMA specific CAR-T cells dosed in MM1.S mouse model and 31% to36% for BCMA specific CAR-T cells dosed in Molp8 mouse model. Anequivalent total dose of untransduced T cells was used for the controlgroup. The control T cell-treated group exhibited progressive tumorgrowth until the study endpoint was reached at day 35 for MM1.S and day23 for Molp8. Statistical analysis of the tumor burden using theRM-ANOVA test with Dunnets correction showed that in all three BCMAspecific CAR-T treated groups, tumor burden was significantly lowercompared to the tumor burden in the control group (p<0.01) (Tables 11and 12). For example, in the MM1.S tumor model, mean total flux inanimals treated with P5AC1-V2 BCMA specific CAR-T cells was 6.44 log10photons/s at day 25, compared to 9.22 log10 photons/s in animals givencontrol T cells (Table 11). At day 35 post tumor implantation, meantotal flux in animals treated with P5AC1-V2 BCMA specific CAR-T cellswas 6.82 log10 photons/s, compared to 10.18 log10 photons/s in animalsgiven control T cells (Table 11). In the Molp8 tumor model, mean totalflux in animals treated with P5AC1-V2 BCMA specific CAR-T cells was 7.88log10 photons/s at day 14, compared to 9.39 log10 photons/s in animalsgiven control T cells (Table 12). At day 23 post tumor implantation,mean total flux in animals treated with P5AC1-V2 BCMA specific CAR-Tcells was 9.29 log10 photons/s, compared 10.37 log10 photons/s inanimals given control T cells (Table 12).

These results demonstrate that treatments with BCMA specific CAR-T cellsare effective to induce tumor regression.

TABLE 11 Tumor bioluminescence measurements of orthotopic MM1.S tumormodel Group 1: Control T cells Days after tumor Mean total fluximplantation (log10 photons/s) SEM N 17 7.84 0.04 10 21 8.16 0.19 10 259.22 0.02 10 28 9.53 0.02 10 32 9.96 0.05 10 35 10.18 0.07 10 Days aftertumor Mean total flux implantation (log10) SEM N Group 2: P5AC1-V2 BCMAspecific CAR-T cells 17 7.84 0.03 10 21 8.14 0.11 10 25 6.44 0.16 10 286.51 0.09 10 32 6.72 0.10 10 35 6.82 0.09 10 Group 3: PC1C12-V2 BCMAspecific CAR-T cells 17 7.86 0.04 10 21 8.56 0.15 10 25 6.85 0.26 10 286.41 0.30 10 32 6.64 0.29 10 35 6.62 0.30 10 Group 4: COM22-V2 BCMAspecific CAR-T cells 17 7.84 0.04 10 21 8.49 0.10 10 25 6.55 0.08 10 286.40 0.09 10 32 6.98 0.14 10 35 6.87 0.22 10

TABLE 12 Tumor bioluminescence measurements of orthotopic Molp-8 tumormodel Days after tumor Mean total flux implantation (log10) SEM N Group1: T cell only control 0 5.80 0.02 10 7 7.48 0.04 10 10 8.24 0.06 10 149.39 0.04 10 17 9.88 0.03 10 21 10.12 0.04 10 23 10.37 0.03 10 Group 2:P5AC1-V2 BCMA specific CAR-T cells 0 5.80 0.02 10 7 7.48 0.04 10 10 8.410.05 10 14 7.88 0.18 10 17 7.39 0.21 10 21 7.98 0.12 10 23 8.29 0.11 10Group 3: PC1C12-V2 BCMA specific CAR-T cells 0 5.80 0.02 10 7 7.51 0.0410 10 8.31 0.07 10 14 7.07 0.21 10 17 6.51 0.15 10 21 7.37 0.13 10 237.75 0.13 10 Group 4: COM22-V2 BCMA specific CAR-T cells Days aftertumor Mean total flux implantation (log10 SEM N 0 5.80 0.02 10 7 7.490.04 10 10 8.39 0.07 10 14 7.78 0.16 10 17 7.51 0.21 10 21 7.89 0.17 1023 8.32 0.14 10

Example 6 Treatment of Multiple Myeloma with TCRα/dCK Knockout BCMASpecific CAR-T Cells

This example illustrates the therapeutic activity of BCMA specific CAR-Tcells in orthotopic mouse models of multiple myeloma.

A humanized mouse model was used to evaluate the efficacy of BCMA CAR-Tcells against human myeloma cell lines expressing BCMA. 6 to 8 week oldfemale Nod/Scid IL2rg−/− (NSG) mice were purchased from the JacksonLaboratories. All animals were housed in a pathogen free vivariumfacility at Rinat and experiments were conducted according to theprotocols in accordance with the Institutional Animal Care and UseCommittee (IACUC) guidelines.

The MM1.S cell lines was purchased from the American Type CultureCollection (ATCC.org). The Cell line was engineered to express a Luc-GFPfusion protein using lentiviral particles (amsbio)and gene edited usingTALEN nucleases to disable the deoxycytidine (dCK) gene. Cells werecultured in RPMI 1640 medium with L-glutamine supplemented with 10%fetal calf serum at 37° C. in 5% carbon dioxide (CO₂). Cells growing inan exponential growth phase were harvested and used for tumorinoculation.

Therapeutic CAR-T cells were produced as described. Healthy human donorcells, peripheral blood mononuclear cells (PBMC) or purified pan-Tcells, are activated and transduced with lentiviral particles encodingfor BCMA scFV, CD8 hinge, CD8 transmembrane, 41BB and CD3 with RQR8genes under the control of the EF-1a promoter. The BCMA specific CAR-Tcells were gene edited to delete the TCRα and/or the dCK gene using acombination of TCRα and dCK TALEN, or TCRα TALEN alone. Transductionefficiency for all T cells was 70%. TCRα knockout T cells were purifiedusing magnetic selection kits for CD3-positive cells (Miltenyi); dCKknockout T cells were purified by expansion in the presence of 0.5 μMclofarabine. Cells were cultured for 14 to 17 days and thencryopreserved in 90% FCS/10% DMSO. For T cell injection, T cells wererapidly thawed in a 37° C. water bath and washed twice with RPMI 1640medium containing 25mM Hepes. For treatment, T cells were injected in0.2 ml RPMI 1640 with 25 mM Hepes into the tail vein of tumor-bearinganimals.

For the mouse tumor model, animals were injected with MM1.S/dCK KO tumorcells. Mice were then treated with 2.5×10⁶ BCMA specific CAR-T cells onday 18 post tumor cell implantation. An equivalent dose of untransducedT cells that received TCRα and dCK TALEN was used as control. Animalswere treated with clofarabine or vehicle for five days after T cellinjection.

Results: the control T cell-treated group exhibited progressive tumorgrowth until the study endpoint was reached at day 35 (Table 13, Group1). Compared against control, groups treated with TCRα knockout BCMAspecific CAR-T cells and vehicle exhibited a significant decrease intumor burden (p<0.05) that was diminished upon coadministration ofclofarabine (p<0.05) (Table 13, Groups 2 and 3). Tumor burden wassignificantly reduced in animals treated with TCRα/dCK double knockoutCAR-T cells, irrespective of whether the animals received vehicle orclofarabine (p<0.05) (Table 13, Groups 4 and 5). Reduction of tumorburden in the groups receiving TCRα/dCK double knockout T cells did notdiffer from the group receiving TCRα single knockout T cells and vehicle(p>0.1) (Table 13, Groups 2, 4, and 5).

These results demonstrate that treatments with TCRα/dCK double knockoutBCMA CAR-T cells are effective to induce tumor regression in thepresence of nucleoside analog therapies such as fludarabine andclofarabine.

TABLE 13 Tumor bioluminescence measurements of nucleoside analogtherapy-resistant orthotopic MM1.S tumor model. Group 1: TCRα/dCK KOcontrol T cells + clofarabine Days after T cell Mean total fluxadministration (log10 photons/s) SEM N 0 7.87 0.04 10 4 8.94 0.08 10 89.22 0.05 10 11 9.52 0.04 10 15 10.00 0.04 10 18 10.38 0.04 10 Daysafter T cell Mean total flux administration (log10) SEM N Group 2: TCRαKO BCMA specific CAR-T cells + vehicle 0 7.86 0.04 10 4 9.28 0.07 10 88.58 0.12 10 11 8.04 0.14 10 15 8.14 0.15 10 18 8.24 0.15 10 Group 3:TCRα KO BCMA specific CAR-T cells + clofarabine 0 7.87 0.04 10 4 9.330.07 10 8 9.17 0.07 10 11 8.95 0.14 10 15 9.36 0.08 10 18 9.50 0.07 10Group 4: TCRα/dCK KO BCMA specific CAR-T cells + vehicle 0 7.86 0.04 104 9.19 0.08 10 8 9.08 0.12 10 11 8.59 0.18 10 15 8.60 0.21 10 18 8.690.18 10 Group 5: TCRα/dCK KO BCMA specific CAR-T cells + clofarabine 07.87 0.04 10 4 9.26 0.09 10 8 9.07 0.10 10 11 8.51 0.14 10 15 8.42 0.2110 18 8.49 0.18 10

Although the disclosed teachings have been described with reference tovarious applications, methods, kits, and compositions, it will beappreciated that various changes and modifications can be made withoutdeparting from the teachings herein and the claimed invention below. Theforegoing examples are provided to better illustrate the disclosedteachings and are not intended to limit the scope of the teachingspresented herein. While the present teachings have been described interms of these exemplary embodiments, the skilled artisan will readilyunderstand that numerous variations and modifications of these exemplaryembodiments are possible without undue experimentation. All suchvariations and modifications are within the scope of the currentteachings.

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

The foregoing description and Examples detail certain specificembodiments of the invention and describes the best mode contemplated bythe inventors. It will be appreciated, however, that no matter howdetailed the foregoing may appear in text, the invention may bepracticed in many ways and the invention should be construed inaccordance with the appended claims and any equivalents thereof.

1-36. (canceled)
 37. An engineered immune cell expressing at its cellsurface membrane a B-cell maturation antigen (BCMA) specific chimericantigen receptor (CAR) comprising an extracellular ligand-bindingdomain, a first transmembrane domain, and an intracellular signalingdomain, wherein the extracellular domain comprises a single chain Fvfragment (scFv) comprising a heavy chain variable (VH) region comprisingthree complementarity determining regions (CDRs) comprising thesequences shown in SEQ ID NO: 33, 72, 39, 76, 83, 92, 25, 112, or 8 ofTable 1; and a light chain variable (VL) region comprising three CDRscomprising the sequences shown in SEQ ID NO: 34, 73, 40, 77, 84, 93, 18,38, or 80 of Table 1, wherein the first transmembrane domain comprises aCD8α chain transmembrane domain, and wherein the intracellular signalingdomain comprises a CD3ζ signaling domain and/or a 4-1BB signalingdomain.
 38. The engineered immune cell of claim 37, wherein the VHregion of the BCMA specific CAR comprises a VH CDR1 comprising the aminoacid sequence shown in SEQ ID NO: 150, 151, or 152; a VH CDR2 comprisingthe amino acid sequence shown in SEQ ID NO: 153 or 154; and a VH CDR3comprising the amino acid sequence shown in SEQ ID NO: 155; and the VLregion of the BCMA specific CAR comprises a VL CDR1 comprising the aminoacid sequence shown in SEQ ID NO: 209; a VL CDR2 comprising the aminoacid sequence shown in SEQ ID NO: 221; and a VL CDR3 comprising theamino acid sequence shown in SEQ ID NO:
 222. 39. The engineered immunecell of claim 38, wherein the VH region comprises the amino acidsequence shown in SEQ ID NO: 33 and the VL region comprises the aminoacid sequence shown in SEQ ID NO:
 34. 40. The engineered immune cell ofclaim 37, wherein the VH region of the BCMA specific CAR comprises a VHCDR1 comprising the amino acid sequence shown in SEQ ID NO: 151, 156, or157; a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:158 or 159; and a VH CDR3 comprising the amino acid sequence shown inSEQ ID NO: 155; and wherein the VL region of the BCMA specific CARcomprises a VL CDR1 comprising the amino acid sequence shown in SEQ IDNO: 209; a VL CDR2 comprising the amino acid sequence shown in SEQ IDNO: 221; and a VL CDR3 comprising the amino acid sequence shown in SEQID NO:
 225. 41. The engineered immune cell of claim 40, wherein the VHregion comprises the amino acid sequence shown in SEQ ID NO: 112 and theVL region comprises the amino acid sequence shown in SEQ ID NO:
 38. 42.The engineered immune cell of claim 37, wherein the BCMA specific CARcomprises the amino acid sequence shown in SEQ ID NO:
 344. 43. Theengineered immune cell of claim 42, wherein the BCMA specific CARcomprises a CD20 epitope.
 44. The engineered immune cell of claim 43,wherein the CD20 epitope comprises the amino acid sequence shown in SEQID NO: 397 or SEQ ID NO:
 398. 45. The engineered immune cell of claim37, wherein the BCMA specific CAR comprises a CD8α signal peptide havingthe sequence of SEQ ID NO: 318; a VH region having the sequence of SEQID NO: 33; a GS linker having the sequence of SEQ ID NO: 333; a VLregion having the sequence of SEQ ID NO: 34; a CD20 epitope having thesequence of SEQ ID NO: 398; a CD8α hinge having the sequence of SEQ IDNO: 320; a CD8α transmembrane domain having the sequence of SEQ ID NO:322; a 4-1BB intracellular signaling domain having the sequence of SEQID NO: 323; and a CD3ζ intracellular signaling domain having thesequence of SEQ ID NO:
 324. 46. The engineered immune cell of claim 37,wherein the BCMA specific CAR comprises a CD8α signal peptide having thesequence of SEQ ID NO: 318; a VH region having the sequence of SEQ IDNO: 112; a GS linker having the sequence of SEQ ID NO: 333; a VL regionhaving the sequence of SEQ ID NO: 38; a CD20 epitope having the sequenceof SEQ ID NO: 398; a CD8α hinge having the sequence of SEQ ID NO: 320; aCD8α transmembrane domain having the sequence of SEQ ID NO: 322; a 4-1BBintracellular signaling domain having the sequence of SEQ ID NO: 323;and a CD3t intracellular signaling domain having the sequence of SEQ IDNO:
 324. 47. The engineered immune cell of claim 37, wherein the BCMAspecific CAR comprises a CD8α signal peptide having the sequence of SEQID NO: 318; a VH region having the sequence of SEQ ID NO: 112; a GSlinker having the sequence of SEQ ID NO: 333; a VL region having thesequence of SEQ ID NO: 38; a CD8α hinge having the sequence of SEQ IDNO: 320; a CD8α transmembrane domain having the sequence of SEQ ID NO:322; a 4-1BB intracellular signaling domain having the sequence of SEQID NO: 323; and a CD3ζ intracellular signaling domain having thesequence of SEQ ID NO:
 324. 48. The engineered immune cell of claim 37,wherein the BCMA specific CAR further comprises a stalk domain betweenthe extracellular ligand-binding domain and the first transmembranedomain.
 49. The engineered immune cell of claim 48, wherein the stalkdomain is selected from the group consisting of: a human CD8α hinge, anIgG1 hinge, and an FcγRIIIα hinge.
 50. The engineered immune cell ofclaim 37, wherein the BCMA specific CAR further comprises a CD20epitope.
 51. The engineered immune cell of claim 50, wherein the CD20epitope comprises the amino acid sequence shown in SEQ ID NO: 397 or SEQID NO:
 398. 52. The engineered immune cell of claim 37, wherein the BCMAspecific CAR further comprises another extracellular ligand-bindingdomain that is not specific for BCMA binding.
 53. The engineered immunecell of claim 37, wherein the BCMA specific CAR further comprises asecond transmembrane domain, wherein the first transmembrane domain andthe extracellular ligand-binding domain(s) are on a first polypeptide,and wherein the second transmembrane domain and the intracellularsignaling domain(s) are on a second polypeptide, wherein the firsttransmembrane domain comprises a transmembrane domain from the a chainof the high-affinity IgE receptor (FcεRI) and the second transmembranedomain comprises a transmembrane domain from the γ or β chain of FcεRI.54. The engineered immune cell of claim 37, further comprising anotherCAR that is not specific for BCMA.
 55. The engineered immune cell ofclaim 37, further comprising a polynucleotide encoding a suicidepolypeptide.
 56. The engineered immune cell of claim 37, furthercomprising a disruption in one or more endogenous genes, wherein theendogenous gene encodes TCRα, TCRβ, CD52, glucocorticoid receptor (GR),deoxycytidine kinase (dCK), or an immune checkpoint protein such as forexample programmed death-1 (PD-1).
 57. The engineered immune cell ofclaim 37, wherein the immune cell is selected from the group consistingof: a T cell, a dendritic cell, a killer dendritic cell, a mast cell, anNK-cell, and a B cell.
 58. The engineered immune cell of claim 37,wherein the immune cell is an inflammatory T-lymphocyte, a cytotoxicT-lymphocyte, a regulatory T-lymphocyte, or a helper T-lymphocyte. 59.The engineered immune cell of claim 37, wherein the immune cell is anautologous immune cell or an allogeneic immune cell.
 60. Apharmaceutical composition comprising a population of the engineeredimmune cells of claim
 37. 61. A method of treating a subject in needthereof, comprising administering the pharmaceutical composition ofclaim 60 to the subject.
 62. A method of treating a condition associatedwith malignant cells expressing BCMA in a subject in need thereof,comprising administering the pharmaceutical composition of claim 60 tothe subject.
 63. The method of claim 62, wherein the condition is acancer.
 64. The method of claim 63, wherein the cancer is a B-cellrelated cancer selected from the group consisting of multiple myeloma,malignant plasma cell neoplasm, Hodgkin's lymphoma, nodular lymphocytepredominant Hodgkin's lymphoma, Kahler's disease and Myelomatosis,plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia,hairy cell leukemia, B-cell non-Hodgkin's lymphoma (NHL), acute myeloidleukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocyticleukemia (ALL), chronic myeloid leukemia (CML), follicular lymphoma,Burkitt's lymphoma, marginal zone lymphoma, mantle cell lymphoma, largecell lymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia,Waldenstrom's macroglobulienemia, diffuse large B cell lymphoma,follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphatictissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma,Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma,lymphoplasmactyic lymphoma, Waldenström macroglobulinemia, nodalmarginal zone B cell lymphoma, splenic marginal zone lymphoma,intravascular large B-cell lymphoma, primary effusion lymphoma,lymphomatoid granulomatosis, T cell/histiocyte-rich large B-celllymphoma, primary central nervous system lymphoma, primary cutaneousdiffuse large B-cell lymphoma (leg type), EBV positive diffuse largeB-cell lymphoma of the elderly, diffuse large B-cell lymphoma associatedwith inflammation, intravascular large B-cell lymphoma, ALK-positivelarge B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphomaarising in HHV8-associated multicentric Castleman disease, B-celllymphoma unclassified with features intermediate between diffuse largeB-cell lymphoma and Burkitt lymphoma, B-cell lymphoma unclassified withfeatures intermediate between diffuse large B-cell lymphoma andclassical Hodgkin lymphoma, and other B-cell related lymphoma.
 65. Amethod of inhibiting tumor growth or progression in a subject who hasmalignant cells expressing BCMA, comprising administering thepharmaceutical composition of claim 60 to the subject.
 66. A method ofinhibiting metastasis of malignant cells expressing BCMA in a subject inneed thereof, comprising administering the pharmaceutical composition ofclaim 60 to the subject.
 67. The method of claim 62, comprisingadministering a nucleoside analog to the subject.
 68. The method ofclaim 67, wherein the nucleoside analog is fludarabine, cytarabine,and/or clofarabine.
 69. A method of engineering an immune cell, themethod comprising: a. providing an immune cell; and b. introducing intothe cell at least one polynucleotide encoding a B-cell maturationantigen (BCMA) specific chimeric antigen receptor (CAR) comprising anextracellular ligand-binding domain, a first transmembrane domain, andan intracellular signaling domain, wherein the extracellular domaincomprises a single chain Fv fragment (scFv) comprising a heavy chainvariable (VH) region comprising three complementarity determiningregions (CDRs) comprising the sequences shown in SEQ ID NO: 33, 72, 39,76, 83, 92, 25, 112, or 8 of Table 1; and a light chain variable (VL)region comprising three CDRs comprising the sequences shown in SEQ IDNO: 34, 73, 40, 77, 84, 93, 18, 38, or 80 of Table 1, wherein the firsttransmembrane domain comprises a CD8α chain transmembrane domain, andwherein the intracellular signaling domain comprises a CD3ζ signalingdomain and/or a 4-1BB signaling domain.
 70. The method of claim 69,comprising: c. introducing into the cell at least one polynucleotideencoding at least one other CAR which is not specific for BCMA.